Ginkgo biloba levopimaradiene synthase

ABSTRACT

The present invention is directed to nucleic acid sequences of  Ginkgo biloba  diterpene synthases, particularly of a levopimaradiene synthase. More specifically, the invention is directed to a cell of a unicellular organism, such as  Saccharomyces cerevisiae  or  Escherichia coli,  comprising levopimaradiene synthase for the metabolically engineered in vivo biosynthesis of a diterpene and a ginkgolide.

FIELD OF THE INVENTION

The present invention is directed to the fields of molecular biology,molecular genetics, and organic chemistry. Specifically, the presentinvention is directed to the cloning and characterization of at leastone Ginkgo biloba sequence for biosynthesis of ginkgolides. Morespecifically, the present invention is directed to the cloning,characterization and expression of Ginkgo biloba levopimaradienesynthase.

BACKGROUND OF THE INVENTION

The gymnosperm Ginkgo biloba, of the Conopsida class, Ginkgoales order,and Ginkgoaceae family, originated in Eastern China approximately 150million years ago and is the sole living representative of its order(Schwarz and Arigoni, 1999; Benson, L., 1957; Chaw, et al., 2000; Bowe,et al., 2000). This hardy tree, termed a “living fossil” by CharlesDarwin, is well-known for its ability to withstand harsh climateconditions and resist insect infestation (Major, R. T., 1967). Theapparent lack of change over millions of years is presumably due to itslong time span between generations; reproduction begins after 20 yearsof age and continues to 1000 years of age.

G. biloba is renowned as a potent herbal therapeutic that aids in therevascularization of ischemic tissue through improved microcirculation.G. biloba leaf extracts have been used for centuries to treatcerebrovascular and cardiovascular diseases, dementia, tinnitus,arthritis, and vertigo (Itil, et al., 1995; Briskin, D. P., 2000). Thesebeneficial pharmacological effects have been attributed, in part, to theginkgolides, a unique series of diterpene molecules which are highlyspecific platelet-activating factor (PAF) receptor antagonists (Hosfordet al., 1990). Generation of PAF occurs during anaphylaxis or shock andleads to bronchoconstriction, contraction of smooth muscle, and reducedcoronary blood flow, which are often fatal. The isomer known asginkgolide B demonstrates the highest activity of the diterpenes andantagonizes all known PAF-induced membrane events. Furthermore, theAmerican Medical Association recently endorsed the Chinese herb as aviable alternative to traditional approaches in the treatment ofAlzheimer's disease. Recent studies report that the extract delayed theprogression of dementia in approximately one third of the patientsstudied (Le Bars et al., 1997).

Ginkgolides were first isolated from the roots of the Ginkgo tree byFurukawa (1932) and later characterized by K. Nakanishi (1967) andSakabe (1967); the elucidated structures were named Ginkgolides A, B, C,J, and M. In 1967, K. Okabe also established the presence of theginkgolides in the leaves of the Ginkgo tree. Ginkgolides arebiosynthesized from geranylgeranyl diphosphate, the universal diterpeneprecursor. These molecules contain a caged trilactone structure anddisplay a rare tert-butyl group. Analogs are distinguished by the numberand location of hydroxyl group substituents. Recently, the ginkgolidesand bilobalide (a pentanorditerpenoid by-product of ginkgolidebiosynthesis) were determined to have significant antifeedant activitiestoward insect larvae (Schwarz, M., 1994; Matsumoto, et al., 1987).

Geranylgeranyl diphosphate (GGDP) (Schwarz and Arigoni, 1999) employedin ginkgolide biosynthesis is derived from isopentenyl diphosphateformed via the deoxyxylulose pathway. The proposed biosynthesis of theginkgolides is initiated by protonation of GGDP to give labdadienyldiphosphate. Ionization of the allylic diphosphate moiety followed by a1,4-hydrogen shift, methyl migration, and deprotonation yieldslevopimaradiene (Schwarz and Arigoni, 1999). The proposed intramolecularhydrogen shift was also observed in the biosynthesis of Abies grandisabietadiene synthase (AgAS) (Ravn et al., 1998; Ravn et al., 2000).Oxidation of ring C produces abietatriene, which is then transportedfrom the plastid to the cytoplasm. The aromatic hydrocarbon undergoesfurther transformation in the endoplasmic reticulum by cytochromeP450-dependent monooxygenases to produce the ginkgolides (Schwarz andArigoni, 1999) (FIG. 1).

Metabolic regulation studies of diterpene production in G. bilobaseedlings indicate that ginkgolides are produced in the roots and aresubsequently translocated to the leaves. Furthermore, diterpenehydrocarbon precursors were found exclusively in the roots and includedlevopimaradiene, palustradiene, abietadiene, pimaradiene, andabietatriene. Addition of cytochrome P450-dependent oxygenase inhibitorsto the roots of seedlings resulted in full inhibition of oxygenationreactions along the pathway to the diterpenes. Abietatriene, the solediterpene hydrocarbon obtained, was identified as the immediateprecursor to the ginkgolides (Cartayrade et al., 1997; Neau et al.,1997).

Presently, commercial development of the ginkgolides as therapeuticagents has been hampered. Because these diterpenoids contain up to 12stereocenters, 4 contiguous quaternary carbons, and 3 oxacyclic ringsfused to 2 spiro carbocyclic rings, they present a formidable syntheticchallenge. In spite of the topological and stereochemical complexityinherent to the ginkgolides, total syntheses of these unusuallychallenging targets have been achieved. In 1988, the first synthesis of(±)-ginkgolide A (38 steps, <1% overall yield) and (±)-ginkgolide B (35steps, <1% overall yield) were reported (Corey and Ghosh, 1988; Corey etal., 1988). Furthermore, ginkgolide B was converted to ginkgolide A in 6steps and approximately 50% yield. More recently, (±)-ginkgolide B wassynthesized in 26 steps and 3% total yield (Crimmins et al., 1999).Although strategically impressive, these demanding routes requiremultiple transformations resulting in low yields that ultimatelypreclude commercial-scale production of the ginkgolides.

Current commercial ginkgolide production relies exclusively onextraction from Ginkgo trees, which accumulate low levels of thecompound. In addition, the demand for this medicinal plant has increasedat a rate of 26% per annum with 2,000 tons harvested annually (Masood,E., 1997) G. biloba plantations serve as the major source of the herbalextract and provide an average 1 to 7 mg/g dry weight ginkgolide fromyoung trees (Balz, et al., 1999) In an effort to increase diterpenoidcontent, G. biloba seedlings, plants, and trees were treated withmetabolic inhibitors that target key branchpoints in isoprenoidbiosynthesis downstream of GGPP synthesis (Huh, et al., 1993)Presumably, inhibiting GGPP depleting pathways would increase theavailable concentration of GGPP, the natural diterpene substrate.Variable results were obtained with cycloartenol synthase inhibitors,ancymidol and AMO-1618. In contrast, application of fluridone (aninhibitor of carotenoid biosynthesis that blocks phytoene desaturation)yielded up to 78% more ginkgolides.

Extraction of the ginkgolides from G. biloba is known. U.S. Pat. No.5,399,348 refers to a method for preparation of Ginkgo biloba extract inwhich the alkylphenol compounds are separated not by using chlorinatedaliphatic hydrocarbon, but through a process of precipitation,filtration and multi step liquid-liquid-extractions. U.S. Pat. No.5,399,348; U.S. Pat. No. 5,322,688; U.S. Pat. No. 5,389,370; U.S. Pat.No. 5,389,370; U.S. Pat. No. 5,637,302; U.S. Pat. No. 5,512,286; U.S.Pat. No. 5,399,348; and U.S. Pat. No. 5,389,370 are all directed tovarious methods of preparing a desired Ginkgo biloba extract. U.S. Pat.No. 5,241,084 and U.S. Pat. No. 5,599,950 are directed to methods toconvert ginkgolide C to ginkgolide B.

Seeking an alternative, non-synthetic approach to ginkgolide production,a method to clone and functionally express genes involved in theirbiosynthesis was considered. In 1971, the isoprenoid nature of theginkgolides was precariously, yet correctly, established using 2-¹⁴C MVAincorporation experiments conducted with G. biloba seedlings. Moreover,the researchers proposed that the unique tert-butyl group arose fromS-adenosyl methionine (Nakanishi, et al., 1971). However, a revisedbiogenetic scheme was put forth as a result of NMR product analyses ofisotopically labeled precursors incubated with G. biloba embryos(Schwarz, et al., 1999). During the course of these extensive studies, adichotomy was observed concerning the biosynthesis of IPP by G. biloba.Specifically, formation of isopentenyl pyrophosphate (IPP), an isopreneunit possessing a diphosphate moiety, proceeds via the classical MVApathway in the synthesis of sitosterol, but in the plastids, thedeoxyxylulose-5-phosphate (DXP) pathway synthesizes GGPP. Presumably,segregation between the two pathways is due to compartmentalization ofthe plant cell. IPP responsible for sitosterol formation is restrictedto the cytoplasm, and IPP incorporated into ginkgolides originates inthe chloroplasts.

G. biloba cultures were first established in 1971; however, HPLCanalysis failed to detect ginkgolides (Nakanishi, et al., 1971). Twodecades later, ginkgolides A and B were detected in undifferentiatedcell cultures (<20 ng/g dry weight), albeit by a factor of 10⁶ less thanthat obtained from leaves of mature trees (Carrier, et al., 1991;Chauret, et al., 1991). Increased ginkgolide content was demonstrated inprimary callus and cell suspension cultures (˜26% and 47% relative toleaves of mature trees, respectively) were unable to be maintained insecondary cultures (Huh, et al., 1993). Currently, high yield productionof the ginkgolides by in vitro cultures of undifferentiated cells hasnot been achieved (Balz et al., 1999). Transgenic cells were obtainedfrom putative transformed G. biloba embryos but ginkgolide concentrationwas <400 μg/g dry tissue culture (Laurain, et al., 1997). Recently,Dupre et al. (2000) reported a reproducible transformation protocol ofG. biloba by Agrobacterium tumefaciens; however, ginkgolide levels ofthe transformed cells have not been disclosed.

There are examples in the art in which heterologous diterpene synthasesare introduced into and expressed in organisms such as Escherichia coli,particularly for the purpose of characterizing activity of a solubleform of the enzyme in the absence of any plastidial targeting sequence(Hill et al., 1996; Peters et al., 2000; Williams et al., 2000).However, the novel levopimaradiene synthase of the present inventionprovides a solution to a need in the art for methods and compositions toquickly produce large amounts of substantially pure ginkgolides in acost-effective manner, particularly in an organism capable of ahigh-yield ginkgolide-producing system.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a purified and isolatednucleic acid sequence encoding a levopimaradiene synthase.

Another embodiment of the present invention is a purified and isolatednucleic acid sequence comprising, SEQ.ID.NO:1, SEQ.ID.NO:32,SEQ.ID.NO:34, SEQ.ID.NO:36 or SEQ.ID.NO:38.

An additional embodiment is a purified and isolated nucleic acidcomprising SEQ.ID.NO:34. Another embodiment is a purified and isolatednucleic acid comprising SEQ.ID.NO:36.

Another embodiment of the present invention is an expression vectorcomprising an isolated and purified nucleic acid sequence encoding alevopimaradiene synthase under control of a promoter operable in a hostcell. In a specific embodiment, the promoter is an inducible promoter,and preferably GAL1. In another specific embodiment, the nucleic acidsequence comprises SEQ.ID.NO:1, SEQ.ID.NO:32, SEQ.ID.NO:34, SEQ.ID.NO:36or SEQ.ID.NO:38.

In yet another specific embodiment of the present invention, the hostcell is a prokaryote, and preferably Escherichia coli. In anotherspecific embodiment, the host cell is a eukaryote, and in a preferredspecific embodiment the eukaryote is a yeast.

Another embodiment of the present invention is an isolated polypeptidehaving an amino acid sequence of a levopimaradiene synthase.

In another embodiment of the present invention there is an isolatedpolypeptide comprising an amino acid sequence of SEQ.ID.NO:2,SEQ.ID.NO:33, SEQ.ID.NO:35, SEQ.ID.NO:37 or SEQ.ID.NO:39.

Another embodiment of the present invention is an isolated polypeptidecomprising an amino acid sequence of SEQ.ID.NO:35. Further, the presentinvention embodies an isolated polypeptide comprising an amino acidsequence of SEQ.ID.NO:37.

Another embodiment of the present invention is an expression vectorcomprising an isolated polynucleotide sequence encoding a polypeptidehaving an amino acid sequence of a levopimaradiene synthase. In aspecific embodiment, the vector further comprises a promoter operativelylinked to the polynucleotide sequence. In a further specific embodiment,the promoter is an inducible promoter. In a preferred specificembodiment, the inducible promoter is GAL1.

In another embodiment there is an isolated polynucleotide sequenceencoding a polypeptide having an amino acid sequence of SEQ.ID.NO:2,SEQ.ID.NO:33, SEQ.ID.NO:35, SEQ.ID.NO:37 or SEQ.ID.NO:39. In a specificembodiment, the vector further comprises a promoter operatively linkedto the polynucleotide sequence. In a further specific embodiment, thepromoter is an inducible promoter and preferably GAL1.

In another embodiment of the present invention, there is a unicellularorganism comprising a purified and isolated nucleic acid sequenceencoding a levopimaradiene synthase. In a specific embodiment, thenucleic acid sequence comprises SEQ.ID.NO:1, SEQ.ID.NO:32, SEQ.ID.NO:34,SEQ.ID.NO:36 or SEQ.ID.NO:38. In a further specific embodiment, thenucleic acid sequence comprises an expression vector. In yet a furtherspecific embodiment, the expression vector comprises an induciblepromoter. In a preferred specific embodiment, the inducible promoter isGAL1.

In another specific embodiment of the present invention, the nucleicacid sequence encoding the levopimaradiene synthase contains a deletioncorresponding to an N-terminal sequence. In yet another specificembodiment, the organism is Saccharomyces, Escherichia coli, Candidaalbicans or Klyveromyces lactis. In a preferred specific embodiment, theorganism is Escherichia coli. In another preferred specific embodiment,the organism is Saccharomyce cerevisiae.

Another embodiment of the present invention is a yeast host cellcomprising a vector, wherein the vector comprises a purified andisolated nucleic acid sequence comprising SEQ.ID.NO:1, SEQ.ID.NO:32,SEQ.ID.NO:34, SEQ.ID.NO:36, or SEQ.ID.NO:38 wherein said nucleic acidsequence is under control of a promoter operable in the yeast host cell.In a further specific embodiment, the nucleic acid sequence comprises anexpression vector.

Yet another embodiment of the present invention is a yeast host cellcomprising a vector, wherein the vector comprises an isolatedpolynucleotide sequence encoding a polypeptide having an amino acidsequence of SEQ.ID.NO:2, SEQ.ID.NO:33, SEQ.ID.NO:35, SEQ.ID.NO:37 orSEQ.ID.NO:39, wherein expression of the polynucleotide is under controlof a promoter operable in the yeast host cell. In a further specificembodiment, the vector is an expression vector.

In one embodiment of the present invention there is a plant host cell,wherein the cell comprises an isolated and purified nucleic acidsequence comprising SEQ.ID.NO:1, SEQ.ID.NO:32, SEQ.ID.NO:34,SEQ.ID.NO:36 or SEQ.ID.NO:38, under control of a promoter operable inthe yeast host cell. In a specific embodiment, the promoter is aninducible promoter. In another specific embodiment, the plant is Ginkgobiloba.

Another embodiment of the present invention there is a unicellularorganism comprising an isolated polynucleotide sequence encoding apolypeptide having an amino acid sequence of a levopimaradiene synthase.In a specific embodiment the amino acid sequence comprises SEQ.ID.NO:2,SEQ.ID.NO:33, SEQ.ID.NO:35, SEQ.ID.NO:37 or SEQ.ID.NO:39. In anotherspecific embodiment, the polynucleotide sequence contains a deletioncorresponding to an N-terminal sequence of the levopimaradiene synthase.

In a specific embodiment, the unicellular organism is Saccharomyces,Escherichia coli, Candida albicans, or Kluyveromyces lactis. In otherspecific embodiments, the unicellular organism is Saccharomycescerevisiae or Escherichia coli.

In one embodiment of the present invention there is a method ofproducing ginkgolide in a cell, comprising the steps of obtaining a cellcomprising an isolated and purified nucleic acid sequence encoding alevopimaradiene synthase; culturing said cell under conditions whereinthe cell produces ginkgolide; and removing the ginkgolide from theculture of cells. In a specific embodiment, the nucleic acid sequencecomprises SEQ.ID.NO:1, SEQ.ID.NO:32, SEQ.ID.NO:34, SEQ.ID.NO:36 orSEQ.ID.NO:38. In a specific embodiment, the cell is Saccharomycescerevisiae. In another specific embodiment, the cell is Escherichiacoli. In a further specific embodiment, the nucleic acid sequencecomprises an expression vector, wherein the expression vector includesan inducible promoter operatively linked to the levopimaradiene synthasecoding region.

In another embodiment of the present invention there is a method ofproducing levopimaradiene in a cell, comprising the steps of obtaining acell comprising an isolated and purified nucleic acid sequence encodinga levopimaradiene synthase; culturing the cell under conditions whereinthe cell produces levopimaradiene; and removing the levopimaradiene fromthe culture of cells. In a specific embodiment, the nucleic acidsequence comprises SEQ.ID.NO:1, SEQ.ID.NO:32, SEQ.ID.NO:34, SEQ.ID.NO:36or SEQ.ID.NO:38. In a further specific embodiment, the nucleic acidsequence comprises an expression vector, wherein the expression vectorincludes an inducible promoter operatively linked to the levopimaradienesynthase coding region.

In another embodiment of the present invention there is a method ofproducing a ginkgolide in a yeast cell, comprising the steps ofobtaining a cell wherein an isolated and purified nucleic acid sequenceof SEQ.ID.NO:1, SEQ.ID.NO:32, SEQ.ID.NO:34, SEQ.ID.NO:36 or SEQ.ID.NO:38under control of a promoter operable in the yeast cell has been added tothe yeast cell; culturing the cell under conditions wherein the yeastcell produces the ginkgolide; and removing the ginkgolide from theculture of yeast cells. In a specific embodiment, the nucleic acidsequence further comprises an inducible promoter.

In another embodiment of the present invention there is a method ofproducing levopimaradiene in a yeast cell, comprising the steps ofobtaining a yeast cell wherein an isolated and purified nucleic acidsequence of SEQ.ID.NO:1, SEQ.ID.NO:32, SEQ.ID.NO:34, SEQ.ID.NO:36 orSEQ.ID.NO:38 under control of a promoter operable in the yeast cell hasbeen added to the yeast cell; culturing the yeast cell under conditionswherein the yeast cell produces the levopimaradiene; and removing thelevopimaradiene from the culture of yeast cells. In a furtherembodiment, the nucleic acid sequence and the promoter comprise anexpression vector.

In another embodiment of the present invention there is a method ofproducing levopimaradiene in a yeast cell, comprising the steps ofobtaining a yeast cell wherein an isolated polynucleotide sequenceencoding a polypeptide having an amino acid sequence of alevopimaradiene synthase under control of a promoter operable in theyeast cell has been added to the yeast cell; culturing the yeast cellunder conditions wherein the yeast cell produces the levopimaradiene;and removing the levopimaradiene from the culture of yeast cells.

In a specific embodiment, the promoter is an inducible promoter. Inanother specific embodiment, the amino acid sequence comprisesSEQ.ID.NO:2, SEQ.ID.NO:33, SEQ.ID.NO:35, SEQ.ID.NO:37 or SEQ.ID.NO:39.

Another embodiment of the present invention is a method of producinglevopimaradiene in a cell, comprising the steps of obtaining a yeastcell, wherein an isolated and purified nucleic acid sequence ofSEQ.ID.NO:1, SEQ.ID.NO:32, SEQ.ID.NO:34, SEQ.ID.NO:36 or SEQ.ID.NO:38under control of a promoter operable in the yeast cell has been added tothe yeast cell and the yeast cell further comprises an increase in theeffective amount of geranylgeranyl diphosphate; growing a culture of theyeast cells; and removing the levopimaradiene from the culture of yeastcells.

In another embodiment of the present invention, there is a ginkgolide,wherein said ginkgolide is obtained from production in a unicellularorganism comprising a purified and isolated nucleic acid sequenceencoding a levopimaradiene synthase.

In another embodiment of the present invention, there is a ginkgolide,wherein said ginkgolide is obtained from production in a unicellularorganism comprising a purified and isolated nucleic acid sequence ofSEQ.ID.NO:1, SEQ.ID.NO:32, SEQ.ID.NO:34, SEQ.ID.NO:36 or SEQ.ID.NO:38.

Another embodiment of the present invention is a ginkgolide, wherein theginkgolide is obtained from production in a unicellular organismcomprising an expression vector having an isolated and purified nucleicacid sequence encoding a levopimaradiene synthase under control of apromoter operable in the organism.

Another embodiment of the present invention is a ginkgolide, wherein theginkgolide is obtained from production in a unicellular organism,wherein the organism comprises an isolated polynucleotide sequenceencoding a polypeptide having an amino acid sequence of SEQ.ID.NO:2,SEQ.ID.NO:33, SEQ.ID.NO:35, SEQ.ID.NO:37 or SEQ.ID.NO:39.

In another embodiment of the present invention, there is a ginkgolide,wherein said ginkgolide is obtained from the method of producing theginkgolide in a cell comprising the steps of obtaining a culture ofcells wherein at least one cell comprises a purified and isolatednucleic acid sequence encoding a levopimaradiene synthase; culturing thecell under conditions wherein the cell produces the ginkgolide; andremoving the ginkgolide from the culture of cells. In a specificembodiment, the nucleic acid sequence comprises SEQ.ID.NO:1,SEQ.ID.NO:32, SEQ.ID.NO:34, SEQ.ID.NO:36 or SEQ.ID.NO:38.

In another embodiment of the present invention, there is a ginkgolide,wherein said ginkgolide is obtained from the method of producing theginkgolide in a yeast cell, comprising the steps of obtaining a cultureof yeast cells, wherein at least one yeast cell comprises a purified andisolated nucleic acid sequence of SEQ.ID.NO:1, SEQ.ID.NO:32,SEQ.ID.NO:34, SEQ.ID.NO:36 or SEQ.ID.NO:38; culturing the yeast cellunder conditions wherein the yeast cell produces the ginkgolide; andremoving the ginkgolide from the culture of yeast cells.

In another embodiment of the present invention, there is a ginkgolide,wherein said ginkgolide is obtained from production in a unicellularorganism which includes an isolated polynucleotide sequence encoding apolypeptide having an amino acid sequence of a levopimaradiene synthase,wherein the polynucleotide sequence comprises a deletion correspondingto an N-terminal sequence; culturing the cell under conditions whereinthe cell produces the ginkgolide; and removing the ginkgolide from theculture of cells. In a specific embodiment, the amino acid sequencecomprises SEQ.ID.NO:33, SEQ.ID.NO:35, SEQ.ID.NO:37 or SEQ.ID.NO:39.

In an additional embodiment of the present invention, there is a nucleicacid sequence comprising SEQ.ID.NO:5, SEQ.ID.NO:6, SEQ.ID.NO:7,SEQ.ID.NO:8, SEQ.ID.NO:9, SEQ.ID.NO:10, SEQ.ID.NO:11, SEQ.ID.NO:12,SEQ.ID.NO:29, SEQ.ID.NO:30, SEQ.ID.NO:31 or SEQ.ID.NO:40.

In an additional embodiment of the present invention there is atransgenic plant, wherein the plant comprises a purified and isolatednucleic acid sequence encoding a levopimaradiene synthase under controlof a promoter operable in the plant. In a specific embodiment, the plantis Ginkgo biloba. In another specific embodiment, the nucleic acidsequence comprises SEQ.ID.NO:1, SEQ.ID.NO:32, SEQ.ID.NO:34, SEQ.ID.NO:36or SEQ.ID.NO:38.

In another specific embodiment, there is a seed of the transgenic plant.In a preferred embodiment, the seed is Ginkgo biloba.

Other and further objects, features, and advantages are apparent andeventually more readily understood by reading the followingspecification and the accompanying drawings forming a part thereof, orany examples of the presently preferred embodiments of the inventiongiven for the purpose of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein:

FIG. 1 depicts the biosynthesis of ginkgolide A from geranylgeranyldiphosphate (GGDP).

FIG. 2 illustrates the structure of an isoprene unit.

FIG. 3 illustrates the parent ginkgolide chemical structure.

FIG. 4 illustrates amino acid sequence alignment of plant sesquiterpeneand diterpene synthases.

DESCRIPTION OF THE INVENTION

It will be readily apparent to one skilled in the art that variousembodiments and modifications may be made in the invention disclosedherein without departing from the scope and spirit of the invention.

As used in the specification, “a” or “an” may mean one or more. As usedin the claim(s), when used in conjunction with the word “comprising”,the words “a” or “an” may mean one or more than one. As used herein“another” may mean at least a second or more.

The technology of the present invention is related to the inventiondescribed in the U.S. patent application entitled, “Diterpene-ProducingUnicellular Organism” filed on the same day and incorporated byreference herein.

I. Definitions

The term “diterpene” as used herein is defined as a terpene compoundcomprised of four isoprene units to yield a 20-carbon hydrocarbonstructure. The 20 carbon acyclic structure is called geranylgeranylpyrophosphate (GGPP) or equally correct, geranylgeranyl diphosphate(GGDP). A skilled artisan is aware that diterpenes result frommetabolism of GGPP and, thus may, after metabolism, yield a structurepossessing one or more rings, one or more double bonds or one or morehydroxyl group. Non-limiting examples of diterpenes are levopimaradienecopalol, abietadiene and abietatriene.

The term “GGDP” as used herein is defined as geranylgeranyl diphosphate.The term may be used interchangeably with geranylgeranyl pyrophosphate(GGPP).

The term “GGPP” as used herein is defined as geranylgeranylpyrophosphate. The term may be used interchangeably with geranylgeranyldiphosphate (GGDP).

The term “diterpenoid” as used herein is defined as a metabolite of aditerpene. One skilled in the art recognizes that a diterpene is oftenfurther transformed and, thus, may possess in an intermediate or finalstructure, more or less than the starting 20-carbons, one or morefunctional groups such as, for example, an ether, a carbonyl, anhydroxyl group or an aromatic ring.

The term “ginkgolide” as used herein is defined as a diterpenoid fromthe Ginkgo biloba plant. A skilled artisan is aware that there are atleast the following naturally occurring ginkgolides: Ginkgolide A,Ginkgolide B, Ginkgolide C, Ginkgolide M, and Ginkgolide J. A skilledartisan is also aware that there are additionally many derivativesthereof, such as, for example, a ketone (i.e., an acetate) at least oneof any of the R groups in FIG. 3. A skilled artisan is aware thatfunctional groups are often altered on a structure to effectcharacteristics such as, for example, solubility, and is very importantin developing, for example, efficacious pharmaceuticals and medicaments.

The term “gymnosperm” as used herein is defined as a plant whose seedsare not enclosed within an ovary. Gymnosperms are contained in fourphyla: Cycadophyta, Ginkgophyta, Pinophyta, and Gnetophyta. Examplesinclude ginkgo, cycad, yew and conifer. A skilled artisan is aware ofreadily accessible databases that provide a comprehensive list ofspecific examples.

The term “levopimaradiene synthase” as used herein is defined as anenzyme which catalyzes the synthesis of levopimaradiene fromgeranylgeranyl diphosphate through ionization of the allylic diphosphatemoiety of labdadienyl pyrophosphate, followed by 1,4 hydrogen shift,methyl migration, and deprotonation.

II. The Present Invention

Levopimaradiene synthase is useful to produce the ginkgolide precursorlevopimaradiene. Potential levopimaradiene production methods of thepresent invention include in vitro conversion of geranylgeranyldiphosphate (GGDP) and in vivo production (in Ginkgo or microorganisms)using biosynthetic GGDP at native levels or in organisms geneticallymodified to increase the effective amount of geranylgeranyl diphosphatelevels. The increase in the effective amount of GGDP allows moresubstrate (e.g., GGDP) to be available for conversion to levopimaradieneand other enzyme diterpene products without the host organism sufferingadverse consequences of low (i.e., below required levels) GGDP levels.

Levopimaradiene synthase overexpression in Ginkgo in a specificembodiment allows increased levels of more advanced ginkgolideprecursors. In alternative embodiments, additional genes areincorporated for increased quantities of levopimaradiene synthase,thereby leading to increased quantities of levopimaradiene or aginkgolide. Expression of levopimaradiene synthase, which preferablydoes not contain a plastidial targeting sequence (see, for example,Peters et al. (2000); Williams et al. (2000)), in organisms that expressgenes encoding enzymes to metabolize GGDP, whether GGDP is exogenouslyprovided or produced de novo, provide production of ginkgolide orginkgolide precursors. One such ginkgolide precursor is levopimaradiene.

Levopimaradiene synthase, which directs the first committed step inginkgolide biosynthesis, was cloned and characterized to ultimatelyisolate and functionally express genes involved in ginkgolidebiosynthesis. This gene is essential to overproduction of ginkgolideusing genetically modified organisms. A skilled artisan is aware that ifthe synthase exhibits low solubility and expression in Escherichia coli,Saccharomyces cerevisiae or other expression hosts, alternative strainsand/or gene truncations are employed.

Ginkgo biloba levopimaradiene synthase is a cytosolically-synthesizedplastid protein containing an N-terminal sequence that directstranslocation of the levopimaradiene to specific plastidialcompartments. The signal sequence is then excised by a specificprotease, yielding a mature levopimaradiene synthase. The optimaltruncation site is determined through, for example, expression studiesof the full-length gene and truncated versions, as described herein. Thepresent invention contemplates a levopimaradiene synthase nucleic acidsequence and amino acid sequence that contains a deletion in theN-terminal sequence.

A skilled artisan is aware of standard means in the art to identifyother levopimaradiene synthase nucleic acid sequences or other nucleicacid sequences which encode gene products that are functionallyinterchangeable with levopimaradiene synthase, meaning catalyzeproduction of a deterpene, for example by searching publicly availablesequence repositories such as GenBank or commercially available sequencerepositories that are readily available. The SEQ.ID.NO:1 nucleic acidsequence is the Ginkgo biloba levopimaradiene synthase nucleic acidsequence, which encodes the Ginkgo biloba levopimaradiene synthase aminoacid sequence (SEQ.ID.NO:2). A GenBank search with SEQ.ID.NO:1, theGinkgo biloba levopimaradiene synthase nucleic acid sequence, identifiesthe similar sequence Abies grandis abietadiene synthase U50768.1(SEQ.ID.NO:3) that encodes AAB05407 (SEQ.ID.NO:4), which is also in thescope of the present invention. A skilled artisan is aware of otherstandard methods to clone sequences, such as by library screeningthrough hybridization to similar sequences.

Standard methods and reagents in the field of yeast molecular genetics,particularly regarding Saccharomyces cerevisiae, are well known in theart. References for such methods include Methods in Yeast Genetics, 2000Edition: A Cold Spring Harbor Laboratory Course Manual (Burke et al.,2000) and Current Protocols in Molecular Biology, Chapter 13 (Ausubel etal., 1994), both incorporated by reference herein. A skilled artisan isaware that the Saccharomyces species of choice is S. cerevisiae,although there are other known species of the genus Saccharomycesincluding S. italicus, S. oviformis, S. capensis, S. chevalieri, S.douglasii, S. paradoxus, S. cariocanus, S. kudriavzevii, S. mikatae, S.bayanus, and S. pastorianus.

III. Ginkgolides

A ginkgolide is a diterpenoid from the Ginkgo biloba plant. Examplesinclude the following naturally occurring ginkgolides Ginkgolide A,Ginkgolide B, Ginkgolide C, Ginkgolide M, Ginkgolide J, in addition toother derivatives such as a substituent(s) effecting solubility but notcatalytic activity. A skilled artisan is aware of such moieties andmethods to determine effects such as a desired solubility, electronicinteraction, coordination and the other such properties withoutcompromising biological activity. Preferable ginkgolides which aregenerated with the methods and compositions of the present inventioninclude: Ginkgolide A and Ginkgolide B.

FIG. 3 demonstrates a generic ginkgolide structure with non-limitingexamples of substitutents for R₁, R₂, R₃ and R₄ given in the chart.

IV. Nucleic Acid-Based Expression Systems

A. Vectors

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence is inserted for introduction into acell where it is replicated. A nucleic acid sequence is in one instance“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques, which are described in Maniatis et al., 1988 and Ausubel etal., 1994, both incorporated herein by reference.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors, in one instance, contain a variety of“control sequences,” which refer to nucleic acid sequences necessary forthe transcription and possibly translation of an operably linked codingsequence in a particular host organism. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

1. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind such as RNA polymerase and other transcriptionfactors. The phrases “operatively positioned,” “operatively linked,”“under control,” “under control of a promoter operable in” and “undertranscriptional control” mean that a promoter is in a correct functionallocation and/or orientation in relation to a nucleic acid sequence tocontrol transcriptional initiation and/or expression of that sequence. Apromoter may or may not be used in conjunction with an “enhancer,” whichrefers to a cis-acting regulatory sequence involved in thetranscriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter is referredto as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages are gainedby positioning the coding nucleic acid segment under the control of arecombinant or heterologous promoter, which refers to a promoter that isnot normally associated with a nucleic acid sequence in its naturalenvironment. A recombinant or heterologous enhancer refers also to anenhancer not normally associated with a nucleic acid sequence in itsnatural environment. Such promoters or enhancers may include promotersor enhancers of other genes, and promoters or enhancers isolated fromany other prokaryotic, viral, or eukaryotic cell, and promoters orenhancers not “naturally occurring,” i.e., containing different elementsof different transcriptional regulatory regions, and/or mutations thatalter expression. In addition to producing nucleic acid sequences ofpromoters and enhancers synthetically, sequences may be produced usingrecombinant cloning and/or nucleic acid amplification technology,including PCR™, in connection with the compositions disclosed herein(see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906, each incorporatedherein by reference). Furthermore, it is contemplated the controlsequences that direct transcription and/or expression of sequenceswithin non-nuclear organelles such as mitochondria, chloroplasts, andthe like, is be employed as well.

Naturally, it is important to employ a promoter and/or enhancer thateffectively directs the expression of the DNA segment in the cell type,organelle, and organism chosen for expression. Those of skill in the artof molecular biology generally know the use of promoters, enhancers, andcell type combinations for protein expression, for example, see Sambrooket al. (1989), incorporated herein by reference. The promoters employedmay be constitutive, tissue-specific, inducible, and/or useful under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins and/or peptides. The promoter may be heterologousor endogenous.

Table 1 lists several elements/promoters that may be employed, in thecontext of the present invention, to regulate the expression of a gene.This list is not intended to be exhaustive of all the possible elementsinvolved in the promotion of expression but, merely, to be exemplarythereof. Table 2 provides examples of inducible elements, which areregions of a nucleic acid sequence that is activated in response to aspecific stimulus. TABLE 1 Promoter and/or Enhancer Promoter/EnhancerReferences Immunoglobulin Heavy Chain Banerji et al., 1983; Gilles etal., 1983; Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imleret al., 1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton etal.; 1990 Immunoglobulin Light Chain Queen et al., 1983; Picard et al.,1984 T-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondo etal.; 1990 HLA DQ a and/or DQ β Sullivan et al., 1987 β-InterferonGoodbourn et al., 1986; Fujita et al., 1987; Goodbourn et al., 1988Interleukin-2 Greene et al., 1989 Interleukin-2 Receptor Greene et al.,1989; Lin et al., 1990 MHC Class II 5 Koch et al., 1989 MHC Class IIHLA-DRa Sherman et al., 1989 β-Actin Kawamoto et al., 1988; Ng et al.;1989 Muscle Creatine Kinase (MCK) Jaynes et al., 1988; Horlick et al.,1989; Johnson et al., 1989 Prealbumin (Transthyretin) Costa et al., 1988Elastase I Omitz et al., 1987 Metallothionein (MTII) Karin et al., 1987;Culotta et al., 1989 Collagenase Pinkert et al., 1987; Angel et al.,1987 Albumin Pinkert et al., 1987; Tronche et al., 1989, 1990α-Fetoprotein Godbout et al., 1988; Campere et al., 1989 t-Globin Bodineet al., 1987; Perez-Stable et al., 1990 β-Globin Trudel et al., 1987c-fos Cohen et al., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985Insulin Edlund et al., 1985 Neural Cell Adhesion Molecule Hirsh et al.,1990 (NCAM) α₁-Antitrypain Latimer et al., 1990 H2B (TH2B) Histone Hwanget al., 1990 Mouse and/or Type I Collagen Ripe et al., 1989Glucose-Regulated Proteins Chang et al., 1989 (GRP94 and GRP78) RatGrowth Hormone Larsen et al., 1986 Human Serum Amyloid A (SAA) Edbrookeet al., 1989 Troponin I (TN I) Yutzey et al., 1989 Platelet-DerivedGrowth Factor Pech et al., 1989 (PDGF) Duchenne Muscular DystrophyKlamut et al., 1990 SV40 Banerji et al., 1981; Moreau et al., 1981;Sleigh et al., 1985; Firak et al., 1986; Herr et al., 1986; Imbra etal., 1986; Kadesch et al., 1986; Wang et al., 1986; Ondek et al., 1987;Kuhl et al., 1987; Schaffner et al., 1988 Polyoma Swartzendruber et al.,1975; Vasseur et al., 1980; Katinka et al., 1980, 1981; Tyndell et al.,1981; Dandolo et al., 1983; de Villiers et al., 1984; Hen et al., 1986;Satake et al., 1988; Campbell and/or Villarreal, 1988 RetrovirusesKriegler et al., 1982, 1983; Levinson et al., 1982; Kriegler et al.,1983, 1984a, b, 1988; Bosze et al., 1986; Miksicek et al., 1986;Celander et al., 1987; Thiesen et al., 1988; Celander et al., 1988; Cholet al., 1988; Reisman et al., 1989 Papilloma Virus Campo et al., 1983;Lusky et al., 1983; Spandidos and/or Wilkie, 1983; Spalholz et al.,1985; Lusky et al., 1986; Cripe et al., 1987; Gloss et al., 1987;Hirochika et al., 1987; Stephens et at., 1987; Glue et al., 1988Hepatitis B Virus Bulla et al., 1986; Jameel et al., 1986; Shaul et al.,1987; Spandau et al., 1988; Vannice et al., 1988 Human ImmunodeficiencyVirus Muesing et al., 1987; Hauber et al., 1988; Jakobovits et al.,1988; Feng et al., 1988; Takebe et al., 1988; Rosen et al., 1988;Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989; Braddocket al., 1989 Cytomegalovirus (CMV) Weber et al., 1984; Boshart et al.,1985; Foecking et al., 1986 Gibbon Ape Leukemia Virus Holbrook et al.,1987; Quinn et al., 1989

TABLE 2 Inducible Elements Element Inducer References MT II PhorbolEster (TFA) Palmiter et al., 1982; Heavy metals Haslinger et al., 1985;Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin etal., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouseGlucocorticoids Huang et al., 1981; Lee et al., mammary 1981; Majors etal., 1983; tumor virus) Chandler et al., 1983; Lee et al., 1984; Pontaet al., 1985; Sakai et al., 1988 β-Interferon poly(rI)x Tavernier etal., 1983 poly(rc) Adenovirus 5 E2 E1A Imperiale et al., 1984Collagenase Phorbol Ester (TPA) Angel et al., 1987a Stromelysin PhorbolEster (TPA) Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al.,1987b Murine MX Gene Interferon, Newcastle Hug et al., 1988 DiseaseVirus GRP78 Gene A23187 Resendez et al., 1988 α-2- IL-6 Kunz et al.,1989 Macroglobulin Vimentin Serum Rittling et al., 1989 MHC Class IInterferon Blanar et al., 1989 Gene H-2κb HSP70 E1A, SV40 Large T Tayloret al., 1989, 1990a, Antigen 1990b Proliferin Phorbol Ester-TPA Mordacqet al., 1989 Tumor Necrosis PMA Hensel et al., 1989 Factor ThyroidThyroid Hormone Chatterjee et al., 1989 Stimulating Hormone α Gene

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart. Examples of such regions include the human LIMK2 gene (Nomoto etal. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), murineepididymal retinoic acid-binding gene (Lareyre et al., 1999), human CD4(Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen (Tsumaki, et al.,1998), D1A dopamine receptor gene (Lee, et al., 1997), insulin-likegrowth factor II (Wu et al., 1997), human platelet endothelial celladhesion molecule-1 (Almendro et al., 1996).

2. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-flame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons are eithernatural or synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picomavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements are, in one instance, linked to heterologous openreading frames. Multiple open reading frames are transcribed together,each separated by an IRES, creating polycistronic messages. By virtue ofthe IRES element, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes are efficiently expressed using asingle promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, herein incorporated by reference).

3. Multiple Cloning Sites

Vectors include, in some instances, a multiple cloning site (MCS), whichis a nucleic acid region that contains multiple restriction enzymesites, any of which are used in conjunction with standard recombinanttechnology to digest the vector. (See Carbonelli et al., 1999, Levensonet al., 1998, and Cocea, 1997, incorporated herein by reference.)“Restriction enzyme digestion” refers to catalytic cleavage of a nucleicacid molecule with an enzyme that functions only at specific locationsin a nucleic acid molecule. Many of these restriction enzymes arecommercially available. Use of such enzymes is widely understood bythose of skill in the art. Frequently, a vector is linearized orfragmented using a restriction enzyme that cuts within the MCS to enableexogenous sequences to be ligated to the vector. “Ligation” refers tothe process of forming phosphodiester bonds between two nucleic acidfragments, which may or may not be contiguous with each other.Techniques involving restriction enzymes and ligation reactions are wellknown to those of skill in the art of recombinant technology.

4. Splicing Sites

Most transcribed eukaryotic RNA molecules undergo RNA splicing to removeintrons from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression. (SeeChandler et al., 1997, herein incorporated by reference.)

5. Polyadenylation Signals

In expression, one typically includes a polyadenylation signal to effectproper polyadenylation of the transcript. The nature of thepolyadenylation signal is not believed to be crucial to the successfulpractice of the invention, and/or any such sequence may be employed.Preferred embodiments include the SV40 polyadenylation signal and/or thebovine growth hormone polyadenylation signal, convenient and/or known tofunction well in various target cells. Also contemplated as an elementof the expression cassette is a transcriptional termination site. Theseelements serve to enhance message levels and/or to minimize read throughfrom the cassette into other sequences.

6. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) is employed ifthe host cell is yeast.

7. Selectable and Screenable Markers

In certain embodiments of the invention, the cells contain nucleic acidconstruct of the present invention, a cell may be identified in vitro orin vivo by including a marker in the expression vector. Such markerswould confer an identifiable change to the cell permitting easyidentification of cells containing the expression vector. Generally, aselectable marker is one that confers a property that allows forselection. A positive selectable marker is one in which the presence ofthe marker allows for its selection, while a negative selectable markeris one in which its presence prevents its selection. An example of apositive selectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product, such as a levopimaradienesynthase. Further examples of selectable and screenable markers are wellknown to one of skill in the art, such as amino acid markers including,but not limited to, uracil, leucine, tryptophan and histidinebiosynthetic genes. A host that is auxotrophic for the amino acidbiosynthetic gene used as a selectable marker allows ready screening fortransformer cells comprising the nucleic acid sequence of interest.

B. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these term also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organism that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell is, inmost instances, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny.

Host cells may be derived from prokaryotes or eukaryotes, depending uponwhether the desired result is replication of the vector or expression ofpart or all of the vector-encoded nucleic acid sequences. Numerous celllines and cultures are available for use as a host cell, and they areobtained through, for example, the American Type Culture Collection(ATCC), which is an organization that serves as an archive for livingcultures and genetic materials. An appropriate host is determined by oneof skill in the art based on the vector backbone and the desired result.A plasmid or cosmid, for example, is introduced into a prokaryote hostcell for replication of many vectors. Bacterial cells used as host cellsfor vector replication and/or expression include DH5α, JM109, and KC8,as well as a number of commercially available bacterial hosts such asSURE® Competent Cells and SOLOPACK™ Gold Cells (STRATAGENE®, La Jolla).Alternatively, bacterial cells such as E. coli LE392 are used as hostcells for phage viruses.

Examples of eukaryotic host cells for replication and/or expression of avector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Manyhost cells from various cell types and organisms are available and wouldbe known to one of skill in the art. Similarly, a viral vector may beused in conjunction with either a eukaryotic or prokaryotic host cell,particularly one that is permissive for replication or expression of thevector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

Another such host cell is a cell that accumulates an increase in theamount of geranylgeranyl diphosphate that is biosynthesized de novo. Anexample of such a microorganism is described in co-pending application“Diterpene-producing unicellular organism”, filed on the same day as theinstant application. The increase in the amount of substrate forlevopimaradiene synthase (e.g., geranylgeranyl diphosphate) allows aproportional increase in levopimaradiene production.

C. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems are employed for use with the present invention to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated byreference, and which are bought, for example, under the name MAXBAC® 2.0from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROMCLONTECH™.

Other examples of expression systems include STRATAGENE®'s COMPLETECONTROL™ Inducible Mammalian Expression System, which involves asynthetic ecdysone-inducible receptor, or its E. coli pET BacterialExpression System. Another example of an inducible expression system isavailable from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

V. Nucleic Acid Detection

In addition to their use in directing the expression of levopimaradienesynthase proteins, polypeptides and/or peptides, the nucleic acidsequences disclosed herein have a variety of other uses. For example,they have utility as probes or primers for embodiments involving nucleicacid hybridization.

A. Hybridization

The use of a probe or primer of between 13 and 100 nucleotides,preferably between 17 and 100 nucleotides in length, or in some aspectsof the invention up to 1-2 kilobases or more in length, allows theformation of a duplex molecule that is both stable and selective.Molecules having complementary sequences over contiguous stretchesgreater than 20 bases in length are generally preferred, to increasestability and/or selectivity of the hybrid molecules obtained. Onegenerally prefers to design nucleic acid molecules for hybridizationhaving one or more complementary sequences of 20 to 30 nucleotides, oreven longer where desired. Such fragments may be readily prepared, forexample, by directly synthesizing the fragment by chemical means or byintroducing selected sequences into recombinant vectors for recombinantproduction.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNAs and/or RNAs or to provide primers for amplification ofDNA or RNA from samples. Depending on the application envisioned, onewould desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of the probe or primers for the targetsequence.

For applications requiring high selectivity, one typically desires toemploy relatively high stringency conditions to form the hybrids. Forexample, relatively low salt and/or high temperature conditions, such asprovided by about 0.02 M to about 0.10 M NaCl at temperatures of about50° C. to about 70° C. Such high stringency conditions tolerate little,if any, mismatch between the probe or primers and the template or targetstrand and would be particularly suitable for isolating specific genesor for detecting specific mRNA transcripts. It is generally appreciatedthat conditions are rendered more stringent by the addition ofincreasing amounts of formamide.

For certain applications, for example, site-directed mutagenesis, it isappreciated that lower stringency conditions are preferred. Under theseconditions, hybridization may occur even though the sequences of thehybridizing strands are not perfectly complementary, but are mismatchedat one or more positions. Conditions may be rendered less stringent byincreasing salt concentration and/or decreasing temperature. Forexample, a medium stringency condition could be provided by about 0.1 to0.25 M NaCl at temperatures of about 37° C. to about 55° C., while a lowstringency condition could be provided by about 0.15 M to about 0.9 Msalt, at temperatures ranging from about 20° C. to about 55° C.Hybridization conditions are readily manipulated depending on thedesired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 MM MgCl₂, 1.0 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C.

In certain embodiments, it is advantageous to employ nucleic acids ofdefined sequences of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of being detected. In preferredembodiments, one may desire to employ a fluorescent label or an enzymetag such as urease, alkaline phosphatase or peroxidase, instead ofradioactive or other environmentally undesirable reagents. In the caseof enzyme tags, colorimetric indicator substrates are known that areemployed to provide a detection means that is visibly orspectrophotometrically detectable, to identify specific hybridizationwith complementary nucleic acid containing samples.

In general, it is envisioned that the probes or primers described hereinare useful as reagents in solution hybridization, as in PCR™, fordetection of expression of corresponding genes, as well as inembodiments employing a solid phase. In embodiments involving a solidphase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to hybridization with selected probes under desiredconditions. The conditions selected depend on the particularcircumstances (depending, for example, on the G+C content, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Optimization of hybridization conditions for theparticular application of interest is well known to those of skill inthe art. After washing of the hybridized molecules to removenon-specifically bound probe molecules, hybridization is detected,and/or quantified, by determining the amount of bound label.Representative solid phase hybridization methods are disclosed in U.S.Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods ofhybridization that may be used in the practice of the present inventionare disclosed in U.S. Pat. Nos. 5,849,481, 5,849,486 and 5,851,772. Therelevant portions of these and other references identified in thissection of the Specification are incorporated herein by reference.

B. Amplification of Nucleic Acids

Nucleic acids used as a template for amplification may be isolated fromcells, tissues or other samples according to standard methodologies(Sambrook et al., 1989). In certain embodiments, analysis is performedon whole cell or tissue homogenates or biological fluid samples withoutsubstantial purification of the template nucleic acid. The nucleic acidmay be genomic DNA or fractionated or whole cell RNA. Where RNA is used,it may be desired to first convert the RNA to a complementary DNA.

The term “primer,” as used herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty and/or thirty base pairs in length, but longersequences are also contemplated. Primers are provide in double-strandedand/or single-stranded form, although the single-stranded form ispreferred.

Pairs of primers designed to selectively hybridize to nucleic acidscorresponding to levopimaradiene synthase are contacted with thetemplate nucleic acid under conditions that permit selectivehybridization. Depending upon the desired application, high stringencyhybridization conditions are selected that only allow hybridization tosequences that are completely complementary to the primers. In otherembodiments, hybridization occurs under reduced stringency to allow foramplification of nucleic acids contain one or more mismatches with theprimer sequences. Once hybridized, the template-primer complex iscontacted with one or more enzymes that facilitate template-dependentnucleic acid synthesis. Multiple rounds of amplification, also referredto as “cycles,” are conducted until a sufficient amount of amplificationproduct is produced.

The amplification product is detected or quantified. In certainapplications, the detection is performed by visual means. Alternatively,the detection involves indirect identification of the product viachemiluminescence, radioactive scintigraphy of incorporated radiolabelor fluorescent label or even via a system using electrical and/orthermal impulse signals (Affymax technology; Bellus, 1994).

A number of template dependent processes are available to amplify theoligonucleotide sequences present in a given template sample. One of thebest known amplification methods is the polymerase chain reaction(referred to as PCR™) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1990, each ofwhich is incorporated herein by reference in their entirety.

A reverse transcriptase PCR™ amplification procedure is performed toquantify the amount of mRNA amplified. Methods of reverse transcribingRNA into cDNA are well known and described in Sambrook et al., 1989.Alternative methods for reverse transcription utilize thermostable DNApolymerases. These methods are described in WO 90/07641. Polymerasechain reaction methodologies are well known in the art. Representativemethods of RT-PCR are described in U.S. Pat. No. 5,882,864.

Another method for amplification is ligase chain reaction (“LCR”),disclosed in European Application No. 320 308, incorporated herein byreference in its entirety. U.S. Pat. No. 4,883,750 describes a methodsimilar to LCR for binding probe pairs to a target sequence. A methodbased on PCR™ and oligonucleotide ligase assay (OLA), disclosed in U.S.Pat. No. 5,912,148, may also be used.

Alternative methods for amplification of target nucleic acid sequencesthat are used in the practice of the present invention are disclosed inU.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497,5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905,5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GB ApplicationNo. 2 202 328, and in PCT Application No. PCT/US89/01025, each of whichis incorporated herein by reference in its entirety.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, isalso used as an amplification method in the present invention. In thismethod, a replicative sequence of RNA that has a region complementary tothat of a target is added to a sample in the presence of an RNApolymerase. The polymerase copies the replicative sequence which thenare detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention (Walker et al., 1992). StrandDisplacement Amplification (SDA), disclosed in U.S. Pat. No. 5,916,779,is another method of carrying out isothermal amplification of nucleicacids which involves multiple rounds of strand displacement andsynthesis, i.e., nick translation.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989; Gingeras et al., PCTApplication WO 88/10315, incorporated herein by reference in theirentirety). Davey et al., European Application No. 329 822 disclose anucleic acid amplification process involving cyclically synthesizingsingle-stranded RNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA),which is used in accordance with the present invention.

Miller et al., PCT Application WO 89/06700 (incorporated herein byreference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoterregion/primer sequence to a target single-stranded DNA (“ssDNA”)followed by transcription of many RNA copies of the sequence. Thisscheme is not cyclic, i.e., new templates are not produced from theresultant RNA transcripts. Other amplification methods include “race”and “one-sided PCR” (Frohman, 1990; Ohara et al., 1989).

C. Detection of Nucleic Acids

Following any amplification, it may be desirable to separate theamplification product from the template and/or the excess primer. In oneembodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (Sambrook et al., 1989). Separated amplification products arecut out and eluted from the gel for further manipulation. Using lowmelting point agarose gels, the separated band is removed by heating thegel, followed by extraction of the nucleic acid.

Separation of nucleic acids may also be effected by chromatographictechniques known in art. There are many kinds of chromatography whichmay be used in the practice of the present invention, includingadsorption, partition, ion-exchange, hydroxylapatite, molecular sieve,reverse-phase, column, paper, thin-layer, and gas chromatography as wellas HPLC.

In certain embodiments, the amplification products are visualized. Atypical visualization method involves staining of a gel with ethidiumbromide and visualization of bands under UV light. Alternatively, if theamplification products are integrally labeled with radio- orfluorometrically-labeled nucleotides, the separated amplificationproducts are exposed to x-ray film or visualized under the appropriateexcitatory spectra.

In one embodiment, following separation of amplification products, alabeled nucleic acid probe is brought into contact with the amplifiedmarker sequence. The probe preferably is conjugated to a chromophore butmay be radiolabeled. In another embodiment, the probe is conjugated to abinding partner, such as an antibody or biotin, or another bindingpartner carrying a detectable moiety.

In particular embodiments, detection is by Southern blotting andhybridization with a labeled probe. The techniques involved in Southernblotting are well known to those of skill in the art. See Sambrook etal., 1989. One example of the foregoing is described in U.S. Pat. No.5,279,721, incorporated by reference herein, which discloses anapparatus and method for the automated electrophoresis and transfer ofnucleic acids. The apparatus permits electrophoresis and blottingwithout external manipulation of the gel and is ideally suited tocarrying out methods according to the present invention.

Other methods of nucleic acid detection that are used in the practice ofthe instant invention are disclosed in U.S. Pat. Nos. 5,840,873,5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726, 5,846,729,5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092, 5,861,244,5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124,5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227,5,932,413 and 5,935,791, each of which is incorporated herein byreference.

D. Other Assays

Other methods for genetic screening are used within the scope of thepresent invention, for example, to detect mutations in genomic DNA, cDNAand/or RNA samples. Methods used to detect point mutations includedenaturing gradient gel electrophoresis (“DGGE”), restriction fragmentlength polymorphism analysis (“RFLP”), chemical or enzymatic cleavagemethods, direct sequencing of target regions amplified by PCR™ (seeabove), single-strand conformation polymorphism analysis (“SSCP”) andother methods well known in the art.

One method of screening for point mutations is based on RNase cleavageof base pair mismatches in RNA/DNA or RNA/RNA heteroduplexes. As usedherein, the term “mismatch” is defined as a region of one or moreunpaired or mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNAor DNA/DNA molecule. This definition thus includes mismatches due toinsertion/deletion mutations, as well as single or multiple base pointmutations.

U.S. Pat. No. 4,946,773 describes an RNase A mismatch cleavage assaythat involves annealing single-stranded DNA or RNA test samples to anRNA probe, and subsequent treatment of the nucleic acid duplexes withRNase A. For the detection of mismatches, the single-stranded productsof the RNase A treatment, electrophoretically separated according tosize, are compared to similarly treated control duplexes. Samplescontaining smaller fragments (cleavage products) not seen in the controlduplex are scored as positive.

Other investigators have described the use of RNase I in mismatchassays. The use of RNase I for mismatch detection is described inliterature from Promega Biotech. Promega markets a kit containing RNaseI that is reported to cleave three out of four known mismatches. Othershave described using the MutS protein or other DNA-repair enzymes fordetection of single-base mismatches.

Alternative methods for detection of deletion, insertion or substitutionmutations that are used in the practice of the present invention aredisclosed in U.S. Pat. Nos. 5,849,483, 5,851,770, 5,866,337, 5,925,525and 5,928,870, each of which is incorporated herein by reference in itsentirety.

VI. Site-Directed Mutagenesis

Structure-guided site-specific mutagenesis (also called site-directedmutagenesis) represents a powerful tool for the dissection andengineering of protein-ligand interactions (Wells, 1996, Braisted etal., 1996). The technique provides for the preparation and testing ofsequence variants by introducing one or more nucleotide sequence changesinto a selected DNA.

Site-specific mutagenesis uses specific oligonucleotide sequences whichencode the DNA sequence of the desired mutation, as well as a sufficientnumber of adjacent, unmodified nucleotides. In this way, a primersequence is provided with sufficient size and complexity to form astable duplex on both sides of the deletion junction being traversed. Aprimer of about 17 to 25 nucleotides in length is preferred, with about5 to 10 residues on both sides of the junction of the sequence beingaltered.

The technique typically employs a bacteriophage vector that exists inboth a single-stranded and double-stranded form. Vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage vectors are commercially available and their use is generally wellknown to those skilled in the art. Double-stranded plasmids are alsoroutinely employed in site-directed mutagenesis, which eliminates thestep of transferring the gene of interest from a phage to a plasmid.

In general, one first obtains a single-stranded vector, or melts twostrands of a double-stranded vector, which includes within its sequencea DNA sequence encoding the desired protein or genetic element. Anoligonucleotide primer bearing the desired mutated sequence,synthetically prepared, is then annealed with the single-stranded DNApreparation, taking into account the degree of mismatch when selectinghybridization conditions. The hybridized product is subjected to DNApolymerizing enzymes such as E. coli polymerase I (Klenow fragment) inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed, wherein one strand encodes the originalnon-mutated sequence, and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate hostcells, such as E. coli cells, and clones are selected that includerecombinant vectors bearing the mutated sequence arrangement.

Comprehensive information on the functional significance and informationcontent of a given residue of protein is best be obtained by saturationmutagenesis in which all 19 amino acid substitutions are examined. Theshortcoming of this approach is that the logistics of multiresiduesaturation mutagenesis are daunting (Warren et al., 1996, Brown et al.,1996; Zeng et al., 1996; Burton and Barbas, 1994; Yelton et al., 1995;Jackson et al., 1995; Short et al., 1995; Wong et al., 1996; Hilton etal., 1996). Hundreds, and possibly even thousands, of site specificmutants must be studied. However, improved techniques make productionand rapid screening of mutants much more straightforward. See also, U.S.Pat. Nos. 5,798,208 and 5,830,650, for a description of “walk-through”mutagenesis.

Other methods of site-directed mutagenesis are disclosed in U.S. Pat.Nos. 5,220,007; 5,284,760; 5,354,670; 5,366,878; 5,389,514; 5,635,377;and 5,789,166.

VII. Levopimaradiene Synthase Nucleic Acids

A. Nucleic Acids and Uses Thereof

Certain aspects of the present invention concern at least onelevopimaradiene synthase nucleic acid. In certain aspects, the at leastone levopimaradiene synthase nucleic acid comprises a wild-type ormutant levopimaradiene synthase nucleic acid. In particular aspects, thelevopimaradiene synthase nucleic acid encodes for at least onetranscribed nucleic acid. In certain aspects, the levopimaradienesynthase nucleic acid comprises at least one transcribed nucleic acid.In particular aspects, the levopimaradiene synthase nucleic acid encodesat least one levopimaradiene synthase protein, polypeptide or peptide,or biologically functional equivalent thereof In other aspects, thelevopimaradiene synthase nucleic acid comprises at least one nucleicacid segment of SEQ.ID.NO:1, or at least one biologically functionalequivalent thereof, for example SEQ.ID.NO:32, SEQ.ID.NO:34,SEQ.ID.NO:36, or SEQ.ID.NO:38.

A skilled artisan is aware that a nucleic acid sequence of the presentinvention may be contained on an episome, such as a plasmid or othervector, or may be on a chromosome of an organism, or both.

The present invention also concerns the isolation or creation of atleast one recombinant construct or at least one recombinant host cellthrough the application of recombinant nucleic acid technology known tothose of skill in the art or as described herein. The recombinantconstruct or host cell may comprise at least one levopimaradienesynthase nucleic acid, and may express at least one levopimaradienesynthase protein, polypeptide or peptide, or at least one biologicallyfunctional equivalent thereof.

As used herein “wild-type” refers to the naturally occurring sequence ofa nucleic acid at a genetic locus in the genome of an organism, andsequences transcribed or translated from such a nucleic acid. Thus, theterm “wild-type” also may refer to the amino acid sequence encoded bythe nucleic acid. As a genetic locus may have more than one sequence oralleles in a population of individuals, the term “wild-type” encompassesall such naturally occurring alleles. As used herein the term“polymorphic” means that variation exists (i.e. two or more allelesexist) at a genetic locus in the individuals of a population. As usedherein “mutant” refers to a change in the sequence of a nucleic acid orits encoded protein, polypeptide or peptide that is the result of thehand of man.

A nucleic acid may be made by any technique known to one of ordinaryskill in the art. Non-limiting examples of synthetic nucleic acid,particularly a synthetic oligonucleotide, include a nucleic acid made byin vitro chemical synthesis using phosphotriester, phosphite orphosphoramidite chemistry and solid phase techniques such as describedin EP 266,032, incorporated herein by reference, or via deoxynucleosideH-phosphonate intermediates as described by Froehler et al., 1986, andU.S. patent Ser. No. 5,705,629, each incorporated herein by reference. Anon-limiting example of enzymatically produced nucleic acid include oneproduced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, eachincorporated herein by reference), or the synthesis of oligonucleotidesdescribed in U.S. Pat. No. 5,645,897, incorporated herein by reference.A non-limiting example of a biologically produced nucleic acid includesrecombinant nucleic acid production in living cells, such as recombinantDNA vector production in bacteria (see for example, Sambrook et al.1989, incorporated herein by reference).

A nucleic acid may be purified on polyacrylamide gels, cesium chloridecentrifugation gradients, or by any other means known to one of ordinaryskill in the art (see for example, Sambrook et al. 1989, incorporatedherein by reference).

The term “nucleic acid” generally refers to at least one molecule orstrand of DNA, RNA or a derivative or mimic thereof, comprising at leastone nucleobase, such as, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g. adenine “A,” guanine “G,” thymine “T”and cytosine “C”) or RNA (e.g. A, G, uracil “U” and C). The term“nucleic acid” encompass the terms “oligonucleotide” and“polynucleotide.” The term “oligonucleotide” refers to at least onemolecule of between about 3 and about 100 nucleobases in length. Theterm “polynucleotide” refers to at least one molecule of greater thanabout 100 nucleobases in length. These definitions generally refer to atleast one single-stranded molecule, but in specific embodiments alsoencompass at least one additional strand that is partially,substantially or fully complementary to the at least one single-strandedmolecule. Thus, a nucleic acid may encompass at least onedouble-stranded molecule or at least one triple-stranded molecule thatcomprises one or more complementary strand(s) or “complement(s)” of aparticular sequence comprising a strand of the molecule. As used herein,a single stranded nucleic acid may be denoted by the prefix “ss”, adouble stranded nucleic acid by the prefix “ds”, and a triple strandednucleic acid by the prefix “ts.”

Thus, the present invention also encompasses at least one nucleic acidthat is complementary to a levopimaradiene synthase nucleic acid. Inparticular embodiments the invention encompasses at least one nucleicacid or nucleic acid segment complementary to the sequence set forth inSEQ.ID.NO:1, SEQ.ID.NO:32, SEQ.ID.NO:34, SEQ.ID.NO:36, and/orSEQ.ID.NO:38. Nucleic acid(s) that are “complementary” or“complement(s)” are those that are capable of base-pairing according tothe standard Watson-Crick, Hoogsteen or reverse Hoogsteen bindingcomplementarity rules. As used herein, the term “complementary” or“complement(s)” also refers to nucleic acid(s) that are substantiallycomplementary, as may be assessed by the same nucleotide comparison setforth above. The term “substantially complementary” refers to a nucleicacid comprising at least one sequence of consecutive nucleobases, orsemiconsecutive nucleobases if one or more nucleobase moieties are notpresent in the molecule, are capable of hybridizing to at least onenucleic acid strand or duplex even if less than all nucleobases do notbase pair with a counterpart nucleobase. In certain embodiments, a“substantially complementary” nucleic acid contains at least onesequence in which about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 77%, about 78%, about 79%, about80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,to about 100%, and any range therein, of the nucleobase sequence iscapable of base-pairing with at least one single or double strandednucleic acid molecule during hybridization. In certain embodiments, theterm “substantially complementary” refers to at least one nucleic acidthat may hybridize to at least one nucleic acid strand or duplex instringent conditions. In certain embodiments, a “partly complementary”nucleic acid comprises at least one sequence that may hybridize in lowstringency conditions to at least one single or double stranded nucleicacid, or contains at least one sequence in which less than about 70% ofthe nucleobase sequence is capable of base-pairing with at least onesingle or double stranded nucleic acid molecule during hybridization.

As used herein, “hybridization”, “hybridizes” or “capable ofhybridizing” is understood to mean the forming of a double or triplestranded molecule or a molecule with partial double or triple strandednature. The term “hybridization”, “hybridize(s)” or “capable ofhybridizing” encompasses the terms “stringent condition(s)” or “highstringency” and the terms “low stringency” or “low stringencycondition(s).”

As used herein “stringent condition(s)” or “high stringency” are thosethat allow hybridization between or within one or more nucleic acidstrand(s) containing complementary sequence(s), but precludeshybridization of random sequences. Stringent conditions tolerate little,if any, mismatch between a nucleic acid and a target strand. Suchconditions are well known to those of ordinary skill in the art, and arepreferred for applications requiring high selectivity. Non-limitingapplications include isolating at least one nucleic acid, such as a geneor nucleic acid segment thereof, or detecting at least one specific mRNAtranscript or nucleic acid segment thereof, and the like.

Stringent conditions may comprise low salt and/or high temperatureconditions, such as provided by about 0.02 M to about 0.15 M NaCl attemperatures of about 50° C. to about 70° C. It is understood that thetemperature and ionic strength of a desired stringency are determined inpart by the length of the particular nucleic acid(s), the length andnucleobase content of the target sequence(s), the charge composition ofthe nucleic acid(s), and to the presence of formamide,tetramethylammonium chloride or other solvent(s) in the hybridizationmixture. It is generally appreciated that conditions may be renderedmore stringent, such as, for example, the addition of increasing amountsof formamide.

It is also understood that these ranges, compositions and conditions forhybridization are mentioned by way of non-limiting example only, andthat the desired stringency for a particular hybridization reaction isoften determined empirically by comparison to one or more positive ornegative controls. Depending on the application envisioned it ispreferred to employ varying conditions of hybridization to achievevarying degrees of selectivity of the nucleic acid(s) towards targetsequence(s). In a non-limiting example, identification or isolation ofrelated target nucleic acid(s) that do not hybridize to a nucleic acidunder stringent conditions may be achieved by hybridization at lowtemperature and/or high ionic strength. Such conditions are termed “lowstringency” or “low stringency conditions”, and non-limiting examples oflow stringency include hybridization performed at about 0.15 M to about0.9 M NaCl at a temperature range of about 20° C. to about 50° C. Ofcourse, it is within the skill of one in the art to further modify thelow or high stringency conditions to suite a particular application.

One or more nucleic acid(s) may comprise, or be composed entirely of, atleast one derivative or mimic of at least one nucleobase, a nucleobaselinker moiety and/or backbone moiety that may be present in a naturallyoccurring nucleic acid. As used herein a “derivative” refers to achemically modified or altered form of a naturally occurring molecule,while the terms “mimic” or “analog” refers to a molecule that may or maynot structurally resemble a naturally occurring molecule, but functionssimilarly to the naturally occurring molecule. As used herein, a“moiety” generally refers to a smaller chemical or molecular componentof a larger chemical or molecular structure, and is encompassed by theterm “molecule.”

As used herein a “nucleobase” refers to a naturally occurringheterocyclic base, such as A, T, G, C or U (“naturally occurringnucleobase(s)”), found in at least one naturally occurring nucleic acid(i.e. DNA and RNA), and their naturally or non-naturally occurringderivatives and mimics. Non-limiting examples of nucleobases includepurines and pyrimidines, as well as derivatives and mimics thereof,which generally forms one or more hydrogen bonds (“anneal” or“hybridize”) with at least one naturally occurring nucleobase in mannerthat may substitute for naturally occurring nucleobase pairing (e.g. thehydrogen bonding between A and T, G and C, and A and U).

Nucleobase, nucleoside and nucleotide mimics or derivatives are wellknown in the art, and have been described in exemplary references suchas, for example, Scheit, Nucleotide Analogs (John Wiley, New York,1980), incorporated herein by reference. “Purine” and “pyrimidine”nucleobases encompass naturally occurring purine and pyrimidinenucleobases and also derivatives and mimics thereof, including but notlimited to, those purines and pyrimidines substituted by one or more ofalkyl, carboxyalkyl, amino, hydroxyl, halogen (i.e. fluoro, chloro,bromo, or iodo), thiol, or alkylthiol wherein the alkyl group comprisesof from about 1, about 2, about 3, about 4, about 5, to about 6 carbonatoms. Non-limiting examples of purines and pyrimidines includedeazapurines, 2,6-diaminopurine, 5-fluorouracil, xanthine, hypoxanthine,8-bromoguanine, 8-chloroguanine, bromothymine, 8-aminoguanine,8-hydroxyguanine, 8-methylguanine, 8-thioguanine, azaguanines,2-aminopurine, 5-ethylcytosine, 5-methylcyosine, 5-bromouracil,5-ethyluracil, 5-iodouracil, 5-chlorouracil, 5-propyluracil, thiouracil,2-methyladenine, methylthioadenine, N,N-diemethyladenine, azaadenines,8-bromoadenine, 8-hydroxyadenine, 6-hydroxyaminopurine, 6-thiopurine,4-(6-aminohexyl/cytosine), and the like. Purine and pyrimidinederivatives and mimics are well known in the art.

As used herein, “nucleoside” refers to an individual chemical unitcomprising a nucleobase covalently attached to a nucleobase linkermoiety. A non-limiting example of a “nucleobase linker moiety” is asugar comprising 5-carbon atoms (a “5-carbon sugar”), including but notlimited to deoxyribose, ribose or arabinose, and derivatives or mimicsof 5-carbon sugars. Non-limiting examples of derivatives or mimics of5-carbon sugars include 2′-fluoro-2′-deoxyribose or carbocyclic sugarswhere a carbon is substituted for the oxygen atom in the sugar ring. Byway of non-limiting example, nucleosides comprising purine (i.e. A andG) or 7-deazapurine nucleobases typically covalently attach the 9position of the purine or 7-deazapurine to the 1′-position of a 5-carbonsugar. In another non-limiting example, nucleosides comprisingpyrimidine nucleobases (i.e. C, T or U) typically covalently attach the1 position of the pyrimidine to 1′-position of a 5-carbon sugar (Kombergand Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).However, other types of covalent attachments of a nucleobase to anucleobase linker moiety are known in the art, and non-limiting examplesare described herein.

As used herein, a “nucleotide” refers to a nucleoside further comprisinga “backbone moiety” generally used for the covalent attachment of one ormore nucleotides to another molecule or to each other to form one ormore nucleic acids. The “backbone moiety” in naturally occurringnucleotides typically comprises a phosphorus moiety, which is covalentlyattached to a 5-carbon sugar. The attachment of the backbone moietytypically occurs at either the 3′- or 5′-position of the 5-carbon sugar.However, other types of attachments are known in the art, particularlywhen the nucleotide comprises derivatives or mimics of a naturallyoccurring 5-carbon sugar or phosphorus moiety, and non-limiting examplesare described herein.

A non-limiting example of a nucleic acid comprising such nucleoside ornucleotide derivatives and mimics is a “polyether nucleic acid”,described in U.S. patent Ser. No. 5,908,845, incorporated herein byreference, wherein one or more nucleobases are linked to chiral carbonatoms in a polyether backbone. Another example of a nucleic acidcomprising nucleoside or nucleotide derivatives or mimics is a “peptidenucleic acid”, also known as a “PNA”, “peptide-based nucleic acidmimics” or “PENAMs”, described in U.S. patent Ser. Nos. 5,786,461,5891,625, 5,773,571, 5,766,855, 5,736,336, 5,719,262, 5,714,331,5,539,082, and WO 92/20702, each of which is incorporated herein byreference. A peptide nucleic acid generally comprises at least onenucleobase and at least one nucleobase linker moiety that is either nota 5-carbon sugar and/or at least one backbone moiety that is not aphosphate backbone moiety. Examples of nucleobase linker moietiesdescribed for PNAs include aza nitrogen atoms, amido and/or ureidotethers (see for example, U.S. Pat. No. 5,539,082). Examples of backbonemoieties described for PNAs include an aminoethylglycine, polyamide,polyethyl, polythioamide, polysulfmamide or polysulfonamide backbonemoiety.

Peptide nucleic acids generally have enhanced sequence specificity,binding properties, and resistance to enzymatic degradation incomparison to molecules such as DNA and RNA (Egholm et al., Nature 1993,365, 566; PCT/EP/01219). In addition, U.S. Pat. Nos. 5,766,855,5,719,262, 5,714,331 and 5,736,336 describe PNAs comprising naturallyand non-naturally occurring nucleobases and alkylamine side chains withfurther improvements in sequence specificity, solubility and bindingaffinity. These properties promote double or triple helix formationbetween a target nucleic acid and the PNA.

U.S. Pat. No. 5,641,625 describes that the binding of a PNA may to atarget sequence has applications the creation of PNA probes tonucleotide sequences, modulating (i.e. enhancing or reducing) geneexpression by binding of a PNA to an expressed nucleotide sequence, andcleavage of specific dsDNA molecules. In certain embodiments, nucleicacid analogues such as one or more peptide nucleic acids may be used toinhibit nucleic acid amplification, such as in PCR, to reduce falsepositives and discriminate between single base mutants, as described inU.S. patent Ser. No. 5,891,625.

U.S. Pat. No. 5,786,461 describes PNAs with amino acid side chainsattached to the PNA backbone to enhance solubility. The neutrality ofthe PNA backbone may contribute to the thermal stability of PNA/DNA andPNA/RNA duplexes by reducing charge repulsion. The melting temperatureof PNA containing duplexes, or temperature at which the strands of theduplex release into single stranded molecules, has been described asless dependent upon salt concentration.

One method for increasing amount of cellular uptake property of PNAs isto attach a lipophilic group. U.S. application Ser. No. 117,363, filedSep. 3, 1993, describes several alkylamino functionalities and their usein the attachment of such pendant groups to oligonucleosides. U.S.application Ser. No. 07/943,516, filed Sep. 11, 1992, and itscorresponding published PCT application WO 94/06815, describe othernovel amine-containing compounds and their incorporation intooligonucleotides for, inter alia, the purposes of enhancing cellularuptake, increasing lipophilicity, causing greater cellular retention andincreasing the distribution of the compound within the cell.

Additional non-limiting examples of nucleosides, nucleotides or nucleicacids comprising 5-carbon sugar and/or backbone moiety derivatives ormimics are well known in the art.

In certain aspect, the present invention concerns at least one nucleicacid that is an isolated nucleic acid. As used herein, the term“isolated nucleic acid” refers to at least one nucleic acid moleculethat has been isolated free of, or is otherwise free of, the bulk of thetotal genomic and transcribed nucleic acids of one or more cells,particularly plant cells, and more particularly Ginkgo biloba cells. Incertain embodiments, “isolated nucleic acid” refers to a nucleic acidthat has been isolated free of, or is otherwise free of, bulk ofcellular components and macromolecules such as lipids, proteins, smallbiological molecules, and the like. As different species may have a RNAor a DNA containing genome, the term “isolated nucleic acid” encompassesboth the terms “isolated DNA” and “isolated RNA”. Thus, the isolatednucleic acid may comprise a RNA or DNA molecule isolated from, orotherwise free of, the bulk of total RNA, DNA or other nucleic acids ofa particular species. As used herein, an isolated nucleic acid isolatedfrom a particular species is referred to as a “species specific nucleicacid.” When designating a nucleic acid isolated from a particularspecies, such as human, such a type of nucleic acid may be identified bythe name of the species. For example, a nucleic acid isolated from oneor more humans would be an “isolated human nucleic acid”, a nucleic acidisolated from Ginkgo biloba would be an “isolated Ginkgo biloba nucleicacid”, and the like.

Of course, more than one copy of an isolated nucleic acid may beisolated from biological material, or produced in vitro, using standardtechniques that are known to those of skill in the art. In particularembodiments, the isolated nucleic acid is capable of expressing aprotein, polypeptide or peptide that has diterpene synthase activity,such as levopimaradiene synthase activity. In other embodiments, theisolated nucleic acid comprises an isolated levopimaradiene synthasegene.

Herein certain embodiments, a “gene” refers to a nucleic acid that istranscribed. As used herein, a “gene segment” is a nucleic acid segmentof a gene. In certain aspects, the gene includes regulatory sequencesinvolved in transcription, or message production or composition. Inparticular embodiments, the gene comprises transcribed sequences thatencode for a protein, polypeptide or peptide. In other particularaspects, the gene comprises a levopimaradiene synthase nucleic acid,and/or encodes a levopimaradiene synthase polypeptide or peptide codingsequences. In keeping with the terminology described herein, an“isolated gene” may comprise transcribed nucleic acid(s), regulatorysequences, coding sequences, or the like, isolated substantially awayfrom other such sequences, such as other naturally occurring genes,regulatory sequences, polypeptide or peptide encoding sequences, etc. Inthis respect, the term “gene” is used for simplicity to refer to anucleic acid comprising a nucleotide sequence that is transcribed, andthe complement thereof. In particular aspects, the transcribednucleotide sequence comprises at least one functional protein,polypeptide and/or peptide encoding unit. As is understood by those inthe art, this function term “gene” includes both genomic sequences, RNAor cDNA sequences or smaller engineered nucleic acid segments, includingnucleic acid segments of a non-transcribed part of a gene, including butnot limited to the non-transcribed promoter or enhancer regions of agene. Smaller engineered gene nucleic acid segments may express, or maybe adapted to express using nucleic acid manipulation technology,proteins, polypeptides, domains, peptides, fusion proteins, mutantsand/or such like.

“Isolated substantially away from other coding sequences” means that thegene of interest, in this case the levopimaradiene synthase gene(s),forms the significant part of the coding region of the nucleic acid, orthat the nucleic acid does not contain large portions ofnaturally-occurring coding nucleic acids, such as large chromosomalfragments, other functional genes, RNA or cDNA coding regions. Ofcourse, this refers to the nucleic acid as originally isolated, and doesnot exclude genes or coding regions later added to the nucleic acid bythe hand of man.

In certain embodiments, the nucleic acid is a nucleic acid segment. Asused herein, the term “nucleic acid segment”, are smaller fragments of anucleic acid, such as for non-limiting example, those that encode onlypart of the levopimaradiene synthase peptide or polypeptide sequence.Thus, a “nucleic acid segment” may comprise any part of thelevopimaradiene synthase gene sequence(s), of from about 2 nucleotidesto the full length of the levopimaradiene synthase peptide orpolypeptide encoding region. In certain embodiments, the “nucleic acidsegment” encompasses the full length levopimaradiene synthase gene(s)sequence. In particular embodiments, the nucleic acid comprises any partof the SEQ.ID.NO:1 sequence(s), of from about 2 nucleotides to the fulllength of the sequence disclosed in SEQ.ID.NO:1.

The nucleic acid(s) of the present invention, regardless of the lengthof the sequence itself, may be combined with other nucleic acidsequences, including but not limited to, promoters, enhancers,polyadenylation signals, restriction enzyme sites, multiple cloningsites, coding segments, and the like, to create one or more nucleic acidconstruct(s). The length overall length may vary considerably betweennucleic acid constructs. Thus, a nucleic acid segment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation or use in the intended recombinant nucleicacid protocol.

In a non-limiting example, one or more nucleic acid constructs may beprepared that include a contiguous stretch of nucleotides identical toor complementary to SEQ.ID.NO:1. A nucleic acid construct may be about3, about 5, about 8, about 10 to about 14, or about 15, about 20, about30, about 40, about 50, about 100, about 200, about 500, about 1,000,about 2,000, about 3,000, about 5,000, about 10,000, about 15,000, about20,000, about 30,000, about 50,000, about 100,000, about 250,000, about500,000, about 750,000, to about 1,000,000 nucleotides in length, aswell as constructs of greater size, up to and including chromosomalsizes (including all intermediate lengths and intermediate ranges),given the advent of nucleic acids constructs such as a yeast artificialchromosome are known to those of ordinary skill in the art. It isreadily understood that “intermediate lengths” and “intermediateranges”, as used herein, means any length or range including or betweenthe quoted values (i.e. all integers including and between such values).Non-limiting examples of intermediate lengths include about 11, about12, about 13, about 16, about 17, about 18, about 19, etc.; about 21,about 22, about 23, etc.; about 31, about 32, etc.; about 51, about 52,about 53, etc.; about 101, about 102, about 103, etc.; about 151, about152, about 153, etc.; about 1,001, about 1002, etc,; about 50,001, about50,002, etc; about 750,001, about 750,002, etc.; about 1,000,001, about1,000,002, etc. Non-limiting examples of intermediate ranges includeabout 3 to about 32, about 150 to about 500,001, about 3,032 to about7,145, about 5,000 to about 15,000, about 20,007 to about 1,000,003,etc.

In particular embodiments, the invention concerns one or morerecombinant vector(s) comprising nucleic acid sequences that encode alevopimaradiene synthase protein, polypeptide or peptide that includeswithin its amino acid sequence a contiguous amino acid sequence inaccordance with, or essentially as set forth in, SEQ.ID.NO:2,corresponding to Ginkgo biloba levopimaradiene synthase. In particularaspects, the recombinant vectors are DNA vectors.

The term “a sequence essentially as set forth in SEQ.ID.NO:2” means thatthe sequence substantially corresponds to a portion of SEQ.ID.NO:2 andhas relatively few amino acids that are not identical to, or abiologically functional equivalent of, the amino acids of SEQ.ID.NO:2.Accordingly, a sequence that has between about 70% and about 80%; ormore preferably, between about 81% and about 90%; or even morepreferably, between about 91% and about 99%; of amino acids that areidentical or functionally equivalent to the amino acids of SEQ.ID.NO:2is a sequence that is “essentially as set forth in SEQ.ID.NO:2” Thus, “asequence essentially as set forth in SEQ.ID.NO:1” encompasses nucleicacids, nucleic acid segments, and genes that comprise part or all of thenucleic acid sequences as set forth in SEQ.ID.NO:1, wherein the sequencethat has between about 70% and about 80%; or more preferably, betweenabout 81% and about 90%; or even more preferably, between about 91% andabout 99%; of amino acids that are identical or functionally equivalentto the nucleic acids of SEQ.ID.NO:1.

The term “biologically functional equivalent” is well understood in theart and is further defined in detail herein. A nucleic acid sequenceencoding a polypeptide that performs an equivalent function to thepolypeptide of amino acid SEQ.ID.NO:2 is a sequence that is a“biologically functional equivalent” protein, polypeptide or peptide.Likewise, the nucleic acid sequence encoding the biologically functionalequivalent polypeptide is also contemplated within the scope of theinvention.

The term “conservatively modified variants” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or conservatively modified variants of theamino acid sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenprotein. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon is altered to any of the correspondingcodons described without altering the encoded polypeptide. Such nucleicacid variations are “silent variations” and represent one species ofconservatively modified variation. Every nucleic acid sequence hereinwhich encodes a polypeptide also describes every possible silentvariation of the nucleic acid. One of ordinary skill recognizes thateach codon in a nucleic acid (except AUG, which is ordinarily the onlycodon for methionine; and UGG, which is ordinarily the only codon fortryptophan) is modified to yield a functionally identical molecule.Accordingly, each silent variation of a nucleic acid which encodes apolypeptide of the present invention is implicit in each describedpolypeptide sequence and incorporated herein by reference.

As to amino acid sequences, one of skill recognizes that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Thus, any number of amino acid residues selected from the group ofintegers consisting of from 1 to 15 is so altered. Thus, for example, 1,2, 3, 4, 5, 7, or 10 alterations are made. Conservatively modifiedvariants typically provide similar biological activity as the unmodifiedpolypeptide sequence from which they are derived. For example, substratespecificity, enzyme activity, or ligand/receptor binding is generally atleast 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the native protein forit's native substrate. Conservative substitution tables providingfunctionally similar amino acids are well known in the art.

In certain other embodiments, the invention concerns at least onerecombinant vector that include within its sequence a nucleic acidsequence essentially as set forth in SEQ.ID.NO:1. In particularembodiments, the recombinant vector comprises DNA sequences that encodeprotein(s), polypeptide(s) or peptide(s) exhibiting levopimaradienesynthase activity.

It also understood that amino acid sequences or nucleic acid sequencesmay include additional residues, such as additional N— or C-terminalamino acids or 5′ or 3′ sequences, or various combinations thereof, andyet still be essentially as set forth in one of the sequences disclosedherein, so long as the sequence meets the criteria set forth above,including the maintenance of biological protein, polypeptide or peptideactivity where expression of a proteinaceous composition is concerned.The addition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ and/or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

Excepting intronic and flanking regions, and allowing for the degeneracyof the genetic code, nucleic acid sequences that have between about 70%and about 79%; or more preferably, between about 80% and about 89%; oreven more particularly, between about 90% and about 99%; of nucleotidesthat are identical to the nucleotides of SEQ.ID.NO:1 are nucleic acidsequences that are “essentially as set forth in SEQ.ID.NO:1”.

It also understood that this invention is not limited to the particularnucleic acid of SEQ.ID.NO:1 or amino acid sequences of SEQ.ID.NO:2.Recombinant vectors and isolated nucleic acid segments may thereforevariously include these coding regions themselves, coding regionsbearing selected alterations or modifications in the basic codingregion, and they may encode larger polypeptides or peptides thatnevertheless include such coding regions or may encode biologicallyfunctional equivalent proteins, polypeptide or peptides that havevariant amino acids sequences.

The nucleic acids of the present invention encompass biologicallyfunctional equivalent levopimaradiene synthase proteins, polypeptides,or peptides. Such sequences may arise as a consequence of codonredundancy or functional equivalency that are known to occur naturallywithin nucleic acid sequences or the proteins, polypeptides or peptidesthus encoded. Alternatively, functionally equivalent proteins,polypeptides or peptides may be created via the application ofrecombinant DNA technology, in which changes in the protein, polypeptideor peptide structure may be engineered, based on considerations of theproperties of the amino acids being exchanged. Changes designed by manmay be introduced, for example, through the application of site-directedmutagenesis techniques as discussed herein below, e.g., to introduceimprovements or alterations to the antigenicity of the protein,polypeptide or peptide, or to test mutants in order to examinelevopimaradiene synthase protein, polypeptide or peptide activity at themolecular level.

Fusion proteins, polypeptides or peptides may be prepared, e.g., wherethe levopimaradiene synthase-coding regions are aligned within the sameexpression unit with other proteins, polypeptides or peptides havingdesired functions. Non-limiting examples of such desired functions ofexpression sequences include purification or immunodetection purposesfor the added expression sequences, e.g., proteinaceous compositionsthat may be purified by affinity chromatography or the enzyme labelingof coding regions, respectively.

Encompassed by the invention are nucleic acid sequences encodingrelatively small peptides or fusion peptides, such as, for example,peptides of from about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22,about 23, about 24, about 25, about 26, about 27, about 28, about 29,about 30, about 31, about 32, about 33, about 34, about 35, about 35,about 36, about 37, about 38, about 39, about 40, about 41, about 42,about 43, about 44, about 45, about 46, about 47, about 48, about 49,about 50, about 51, about 52, about 53, about 54, about 55, about 56,about 57, about 58, about 59, about 60, about 61, about 62, about 63,about 64, about 65, about 66, about 67, about 68, about 69, about 70,about 71, about 72, about 73, about 74, about 75, about 76, about 77,about 78, about 79, about 80, about 81, about 82, about 83, about 84,about 85, about 86, about 87, about 88, about 89, about 90, about 91,about 92, about 93, about 94, about 95, about 96, about 97, about 98,about 99, to about 100 amino acids in length, or more preferably, offrom about 15 to about 30 amino acids in length; as set forth inSEQ.ID.NO:2 and also larger polypeptides up to and including proteinscorresponding to the full-length sequences set forth in SEQ.ID.NO:2.

As used herein an “organism” may be a prokaryote, eukaryote, virus andthe like. As used herein the term “sequence” encompasses both the terms“nucleic acid” and “proteinaceous” or “proteinaceous composition.” Asused herein, the term “proteinaceous composition” encompasses the terms“protein”, “polypeptide” and “peptide.” As used herein “artificialsequence” refers to a sequence of a nucleic acid not derived fromsequence naturally occurring at a genetic locus, as well as the sequenceof any proteins, polypeptides or peptides encoded by such a nucleicacid. A “synthetic sequence”, refers to a nucleic acid or proteinaceouscomposition produced by chemical synthesis in vitro, rather thanenzymatic production in vitro (i.e. an “enzymatically produced”sequence) or biological production in vivo (i.e. a “biologicallyproduced” sequence).

VIII. Methods for Plant Transformation

Suitable methods for plant transformation for use with the currentinvention are believed to include virtually any method by which DNA isintroduced into a cell, such as by direct delivery of DNA such as byPEG-mediated transformation of protoplasts (Omirulleh et al., 1993), bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985), byelectroporation (U.S. Pat. No. 5,384,253, specifically incorporatedherein by reference in its entirety), by agitation with silicon carbidefibers (Kaeppler et al., 1990; U.S. Pat. No. 5,302,523, specificallyincorporated herein by reference in its entirety; and U.S. Pat. No.5,464,765, specifically incorporated herein by reference in itsentirety), by Agrobacterium-mediated transformation (U.S. Pat. No.5,591,616 and U.S. Pat. No. 5,563,055; both specifically incorporatedherein by reference) and by acceleration of DNA coated particles (U.S.Pat. No. 5,550,318; U.S. Pat. No. 5,538,877; and U.S. Pat. No.5,538,880; each specifically incorporated herein by reference in itsentirety), and the like. Through the application of techniques such asthese, maize cells as well as those of virtually any other plant speciesmay be stably transformed, and these cells developed into transgenicplants. In certain embodiments, acceleration methods are preferred andinclude, for example, microprojectile bombardment and the like.

A transgenic plant may require seed propagation, and in such instances,a seed of the transgenic plant embodies the recombinant gene therein. Inthe case of Ginkgo, the genetic content of the seeds is particularlyenriched for ginkgolide production. Thus, the seed of a transgenic plantis characterized by increased amounts of a ginkgolide and is areasonable means to propagate the transgenic plant that is the resourcefor the sought-after ginkgolide.

A. Electroporation

Where one wishes to introduce DNA by means of electroporation, it iscontemplated that the method of Krzyzek et al. (U.S. Pat. No. 5,384,253,incorporated herein by reference in its entirety) is particularlyadvantageous. In this method, certain cell wall-degrading enzymes, suchas pectin-degrading enzymes, are employed to render the target recipientcells more susceptible to transformation by electroporation thanuntreated cells. Alternatively, recipient cells are made moresusceptible to transformation by mechanical wounding.

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wounding in acontrolled manner. Examples of some species which have been transformedby electroporation of intact cells include maize (U.S. Pat. No.5,384,253; Rhodes et al., 1995; D'Halluin et al., 1992), wheat (Zhou etal., 1993), tomato (Hou and Lin, 1996), soybean (Christou et al., 1987)and tobacco (Lee et al., 1989).

One also may employ protoplasts for electroporation transformation ofplants (Bates, 1994; Lazzeri, 1995). For example, the generation oftransgenic soybean plants by electroporation of cotyledon-derivedprotoplasts is described by Dhir and Widholm in Intl. Patent Appl. Publ.No. WO 9217598 (specifically incorporated herein by reference). Otherexamples of species for which protoplast transformation has beendescribed include barley (Lazerri, 1995), sorghum (Battraw et al.,1991), maize (Bhattachaijee et al., 1997), wheat (He et al., 1994) andtomato (Tsukada, 1989).

B. Microprojectile Bombardment

A preferred method for delivering transforming DNA segments to plantcells in accordance with the invention is microprojectile bombardment(U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No.5,610,042; and PCT Application WO 94/09699; each of which isspecifically incorporated herein by reference in its entirety). In thismethod, particles may be coated with nucleic acids and delivered intocells by a propelling force. Exemplary particles include those comprisedof tungsten, platinum, and preferably, gold. It is contemplated that insome instances DNA precipitation onto metal particles would not benecessary for DNA delivery to a recipient cell using microprojectilebombardment. However, it is contemplated that particles may contain DNArather than be coated with DNA. Hence, it is proposed that DNA-coatedparticles may increase the level of DNA delivery via particlebombardment but are not, in and of themselves, necessary.

For the bombardment, cells in suspension are concentrated on filters orsolid culture medium. Alternatively, immature embryos or other targetcells may be arranged on solid culture medium. The cells to be bombardedare positioned at an appropriate distance below the macroprojectilestopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System, whichis used to propel particles coated with DNA or cells through a screen,such as a stainless steel or Nytex screen, onto a filter surface coveredwith monocot plant cells cultured in suspension. The screen dispersesthe particles so that they are not delivered to the recipient cells inlarge aggregates. It is believed that a screen intervening between theprojectile apparatus and the cells to be bombarded reduces the size ofprojectiles aggregate and may contribute to a higher frequency oftransformation by reducing the damage inflicted on the recipient cellsby projectiles that are too large.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species. Examples of species forwhich have been transformed by microprojectile bombardment includemonocot species such as maize (PCT Application WO 95/06128), barley(Ritala et al., 1994; Hensgens et al., 1993), wheat (U.S. Pat. No.5,563,055, specifically incorporated herein by reference in itsentirety), rice (Hensgens et al., 1993), oat (Torbet et al., 1995;Torbet et al., 1998), rye (Hensgens et al., 1993), sugarcane (Bower etal., 1992), and sorghum (Casas et al., 1993; Hagio et al., 1991); aswell as a number of dicots including tobacco (Tomes et al., 1990;Buising and Benbow, 1994), soybean (U.S. Pat. No. 5,322,783,specifically incorporated herein by reference in its entirety),sunflower (Knittel et al. 1994), peanut (Singsit et al., 1997), cotton(McCabe and Martinell, 1993), tomato (VanEck et al. 1995), and legumesin general (U.S. Pat. No. 5,563,055, specifically incorporated herein byreference in its entirety).

C. Agrobacterium-mediated Transformation

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA is introduced intowhole plant tissues, thereby bypassing the need for regeneration of anintact plant from a protoplast. The use of Agrobacterium-mediated plantintegrating vectors to introduce DNA into plant cells is well known inthe art. See, for example, the methods described by Fraley et al.,(1985), Rogers et al., (1987) and U.S. Pat. No. 5,563,055, specificallyincorporated herein by reference in its entirety.

Agrobacterium-mediated transformation is most efficient indicotyledonous plants and is the preferable method for transformation ofdicots, including Arabidopsis, tobacco, tomato, and potato. Indeed,while Agrobacterium-mediated transformation has been routinely used withdicotyledonous plants for a number of years, it has only recently becomeapplicable to monocotyledonous plants. Advances inAgrobacterium-mediated transformation techniques have now made thetechnique applicable to nearly all monocotyledonous plants. For example,Agrobacterium-mediated transformation techniques have now been appliedto rice (Hiei et al., 1997; Zhang et al., 1997; U.S. Pat. No. 5,591,616,specifically incorporated herein by reference in its entirety), wheat(McCormac et al., 1998), barley (Tingay et al., 1997; McCormac et al.,1998), and maize (Ishidia et al., 1996).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described(Rogers et al., 1987) have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes. Inaddition, Agrobacterium containing both armed and disarmed T_(i) genesare used for the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

D. Other Transformation Methods

Transformation of plant protoplasts is achieved using methods based oncalcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Omirulleh et al., 1993; Frommet al., 1986; Uchimiya et al., 1986; Callis et al., 1987; Marcotte etal., 1988).

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of cereals from protoplastshave been described (Fujimara et al., 1985; Toriyama et al., 1986;Yamada et al., 1986; Abdullah et al., 1986; Omirulleh et al., 1993 andU.S. Pat. No. 5,508,184; each specifically incorporated herein byreference in its entirety). Examples of the use of direct uptaketransformation of cereal protoplasts include transformation of rice(Ghosh-Biswas et al., 1994), sorghum (Battraw and Hall, 1991), barley(Lazerri, 1995), oat (Zheng and Edwards, 1990) and maize (Omirulleh etal., 1993).

To transform plant strains that are not successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuesare utilized. For example, regeneration of cereals from immature embryosor explants are effected as described (Vasil, 1989). Also, siliconcarbide fiber-mediated transformation is used with or withoutprotoplasting (Kaeppler, 1990; Kaeppler et al., 1992; U.S. Pat. No.5,563,055, specifically incorporated herein by reference in itsentirety). Transformation with this technique is accomplished byagitating silicon carbide fibers together with cells in a DNA solution.DNA passively enters as the cell are punctured. This technique has beenused successfully with, for example, the monocot cereals maize (PCTApplication WO 95/06128, specifically incorporated herein by referencein its entirety; Thompson, 1995) and rice (Nagatani, 1997).

An embodiment of the present invention is to produce significant amountsof ginkgolide precursors and/or ginkgolide in vivo in Ginkgo ormicroorganisms such as Saccharomyces cerevisiae, Escherichia coli,Candida albicans, and the like. Cell suspension cultures of Ginkgobiloba are known in the art (Balz et al., 1999; Fiehe et al., 2000).

In a preferred embodiment, ginkgolide precursors and/or ginkgolides areproduced in vivo by expressing a nucleic acid sequence which encodesGinkgo biloba levopimaradiene synthase, which is a rate-limiting step inthe ginkgolide biosynthesis. In another preferred embodiment, theexpression is upregulated, or “overexpressed” compared to native levelsin wild type. A skilled artisan is aware how to achieve overexpression,such as by controlling regulation of the Ginkgo bil6ba levopimaradienesynthase with a strong promoter, examples of which are known in the art.In another preferred embodiment, the promoter is an inducible promoter,such as GAL1.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those skilled in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus is considered to constitute preferred modesfor its practice. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes made in thespecific embodiments which are disclosed and maintain a like or similarresult without departing from the concept, spirit and scope of theinvention. More specifically, it is apparent that certain agents thatare both chemically and physiologically related may be substituted forthe agents described herein while the same or similar results would beachieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

Example 1 Methods-Plant Materials, Substrates, and Reagents

Ginkgo biloba “white nut” seeds were purchased from Dynasty Supermarket(Houston, Tex.). The seeds were stored at 4° C. for several days beforesowing. Embryos were cultivated under aseptic conditions in an agarmedium supplemented with D-glucose, L-glutamine, and Heller's salts atroom temperature in the dark for four to six weeks (Schwarz, 1994).Synthesis of geranylgeraniol was performed as indicated in Ruan (1999)and Coates et al. (1978). Synthesis of geranylgeranyl diphosphate wasperformed as indicated by Corey et al. (1972), Davisson et al. (1985),and Davisson et al. (1986). Levopimarol was synthesized from levopimaricacid (Helix Biotech; New Westminster, British Columbia, Canada)according to procedures of Abad et al. (1985), Gigante et al. (1999),and Ayer and Talamas (1988). (6E,10E)-Geranyllinalool and pyridiniumdichlorochromate were obtained from Fluka. All other reagents wereobtained from either Sigma/Aldrich or Fisher Scientific, unlessotherwise noted. Dichloromethane, dimethylformamide, methansulfonylchloride, triethylamine, and toluene were freshly distilled over calciumhydride; tetrahydrofuran was freshly distilled over Na/benzophenone.Ammonium molybdate, ascorbic acid, tetrabutylammonium hydroxide, andcitric acid were from ACROS. HP 20 polyaromatic dianion resin (250-850μm) was purchased from Supelco.

Example 2 Methods-G. Biloba mRNA Isolation, cDNA Library Construction,and Quality Assessment

At active growth, embryonic roots were harvested at 33 days (HSG1) and40 days (HSG2) and snap-frozen in liquid nitrogen. Total RNA wasisolated using a modified protocol for total RNA isolation from pinetrees (Chang et al., 1993) in which an acidic phenol/chloroform (1:1)extraction was included prior to ethanol precipitation. Poly(A)+RNA wasselected with oligo(dT)cellulose (Life Technologies MessageMakerPoly(A)+Syringe Kit) and purified using Sephadex gel chromatography(Boehringer Mannheim Mini Quick Spin RNA Column) according tomanufacturer's instructions. cDNA libraries were prepared using theSuperScript™ Plasmid System for cDNA Synthesis and Plasmid Cloning (LifeTechnologies). cDNA constructs (SalI/Not I) were subcloned into both theE. coli expression vector pSPORT1 and the centromeric yeast shuttlevector pRS316GAL (Liu and Krizek, 1992). The resultant plasmids weretransformed by electroporation into ElectroMax™ DH10B Cells (LifeTechnologies). The number of transformants in each library varied from4.8×10⁵ to 3.2×10⁶ with approximate insert size ranging from 200 to 2600bp.

Complementation experiments were conducted to determine the quality ofthe libraries. G. biloba cDNA library HSG2 (10 μg) in pRS316GAL wastransformed into the auxotrophic S. cerevisiae strain JBY575 (MATaura3-52-trpl-Δ63 leu2-3, 112 his3-Δ200 ade2 Gal⁺) (Corey et al., 1996)using the lithium acetate method (Ito et al., 1983), plated ontosynthetic complete medium lacking uracil and supplemented with 2%glucose and 1.5% agar, and grown at 30° C. A total of 1.6×10⁵ colonieswere screened. Prototrophic clones were selected for growth by replicaplating onto synthetic complete medium lacking leucine, tryptophan, orhistidine and supplemented with 2% galactose and 1.5% agar, andincubated at 30° C. The frequency of complementing cDNA was 1 in every17,778 to 40,000 for LEU2, 1 in every 20,000 to 32,000 for TRP1, and 1in every 22,800 to 32,000 for HIS3.

Example 3 Methods-Levopimaradiene Synthase Gene Cloning

PCR degenerate primers were designed according to sequence similaritybetween gymnosperm terpene synthases Abies grandis abietadiene synthase(SEQ.ID.NO:3), Abies grandis E-α-bisabolene synthase (GenBank AccessionNo. AF006195; SEQ.ID.NO:13 or GenBank Accession No. AF006194;SEQ.ID.NO:14; corresponding to the amino acid sequence in GenBankAccession No. AAC24192.1; SEQ.ID.NO:15 or AAC24191.1; SEQ.ID.NO:16,respectively), Abies grandis δ-selinene synthase (GenBank Accession No.U92266; SEQ.ID.NO:17; corresponding to the amino acid sequence inGenBank Accession No. AAC05727.1; SEQ.ID.NO: 18); Abies grandisγ-humulene synthase (GenBank Accession No. U92267; SEQ.ID.NO:19;corresponding to the amino acid sequence in GenBank Accession No.AAC05728.1; SEQ.ID.NO:20), Abies grandis pinene synthase (GenBankAccession No. U87909; SEQ.ID.NO:21; corresponding to the amino acidsequence in GenBank Accession No. AAB71085.1; SEQ.ID.NO:22); Abiesgrandis (−)-4S-limonene synthase (GenBank Accession No. AF006193;SEQ.ID.NO:23; corresponding to the amino acid sequence in GenBankAccession No. AAB70907.1; SEQ.ID.NO:24); Abies grandis myrcene synthase(GenBank Accession No. U87908; SEQ.ID.NO:25; corresponding to the aminoacid sequence in GenBank Accession No. AAB71084.1; SEQ.ID.NO:26); Abiesgrandis (−)-limonene/(−)-alpha-pinene synthase (agc11) (GenBankAccession No. AF139207; SEQ.ID.NO:27; corresponding to the amino acidsequence in GenBank Accession No. AAF61455.1; SEQ.ID.NO:28); and Taxusbrevifolia taxadiene synthase (SEQ.ID.NO:41).

PCR reactions were conducted on 50 μL scale containing 200 ng cDNA, 5.0μL 10× PC2 buffer (500 mM Tris-HCl, pH=9.1, 160 mM (NH4)2SO4, 35 mMMgCl₂), 4.0 μL 2.5 mM dNTPs, and 5.0 μL (20 pmol/μL) forward and reversedegenerate primers. The program employed a 4 min 95° C. hot-start afterwhich 0.5 μL Taq DNA Polymerase (5.0 Units/μL, Fisher Biotech) was addedto the PCR reaction, followed by 40 cycles with 1 min annealing using atemperature gradient from 68° C. to 48° C. (−0.5° C./cycle), 3 minextension at 72° C., and 45 second denaturation at 95° C. The programwas terminated with a 5 min extension at 72° C. An aliquot of eachreaction (5 μL) was analyzed on 2% agarose gel. The first round of PCRreactions employed the degenerate forward primer HSG1FP(5′-GCNTAYGAYACNGCNTGGGT-3′; SEQ.ID.NO:29). Combination with HSG6RP(5′-GCYTKRTANGTYTTNGTRTC-3′; SEQ.ID.NO:30) resulted in a 1907 bpfragment (HSG97), which was re-amplified, gel purified (QIAGEN),quantitated, and ligated into pGEM-T vector (50 ng/μL, Promega).

Conventional abbreviations are used in the primer sequences, wherein Nis any base, K is G or T, Y is a pyrimidine, and R is a purine. Theremainder of the sequence was obtained with specific primers HSG97.3FP(5′-ATGTGGTGGACTGGCAAGAG-3′; SEQ.ID.NO:5) and HSG97.3RP(5′-TAAAGATCGTCCAGAATAAC-3′; SEQ.ID.NO:6). A 1372 bp segment was excisedwith DraI and BsrG I. The DNA fragment (25 ng) was radiolabeled withα-³²P-dCTP using random oligonucleotide primers to probe 3.0×10⁵colonies (cDNA library HSG2E) by colony hybridization (Ausubel et al.,1999).

A total of 10 colonies were obtained, for which an additional round ofscreening yielded 47 hybridizing colonies. Six colonies wereinvestigated further and restriction enzyme mapping indicated that threeclones corresponded to the size of the expected full-length cDNA.Sequence analysis with forward primer T7 and reverse primer SP6indicated that these genes were putative diterpene cyclases based onhomology to Abies grandis abietadiene synthase. All three clonesexhibited identical 5′ and 3′ ends, therefore, one was selected for genecharacterization.

Primers HSG100.1FP (5′-AACTGCCAGATGGCTCGTGG-3′; SEQ.ID.NO:7) andHSG100.2FP (5′-GGTGGAGTATGCTATAAAGT-3′; SEQ.ID.NO:8) were used alongwith HSG97.3FP to obtain the remaining sequence. Sequence data revealedthat a 2681 bp cDNA (HSG100/pSPORT1) had been cloned, however, theinitiation codon was absent. RNA ligase mediated rapid amplification ofcDNA ends (FirstChoice™ RLM-RACE Kit, Ambion) was employed with outergene specific primer HSG1500GS (5′-CAGAGCCGTCAATTGACGGAATTC-3′;SEQ.ID.NO:9) and inner gene specific primer HSG150IGS(5′-CATCGACGCTTGATTTCGATGTCG-3′; SEQ.ID.NO:10) to obtain the N-terminalsequence. The full-length clone (2705 bp) encoded an 873 amino acid openreading frame of 2622 bp with a predicted molecular weight of 100,289.Sequence alignment using the Clustal method indicated a 60% identity toAbies grandis abietadiene synthase, 46% to Abies grandis bisabolenesynthase, and 41% to Taxus brevifolia taxadiene synthase (FIG. 4).

Sequence alignment in FIG. 4 was prepared with MegAlign (DNAStar,Madison, Wis.) using the Clustal method. Amino acid residues identicalin at least three of the four synthases are shaded; hyphens indicategaps inserted to maximize sequence alignment. Lines indicateaspartate-rich motifs, arrows designate regions targeted by degeneratePCR primers, and arrowheads identify N-terminal cleavage sites.

GbLS is Ginkgo biloba levopimaradiene synthase; AgAs is Abies grandisabietadiene synthase; AgBS is Abies grandis bioabolene synthase; andThTS is Taxus brevifolia taxadiene synthase.

Example 4 Methods-cDNA Expression and Enzymatic Assay

Site-directed mutagenesis of HSG100/pSPORT1 with primers Ala²(5′-TTGCAAAGAGCACCCCAGCCATTTTTTTTGTCGACACCCGGGAATT CCGGACCGGT-3′;SEQ.ID.NO:11), Ser⁶¹ (5′-TGGACGAGTCTCTGCAGCTGACATTTTTTTTTGTCGACCAATTCCATCTCAGCCTT-3′; SEQ.ID.NO:12), Leu⁸⁰(5′-TGATAATCCGCATTAAGCATTTTTTTGTCGACTCCTCCTGTGGAAGCTGAT-3′;SEQ.ID.NO:31), and Phe¹²⁹ (5′-TCGCCCATGGACTGAAACATTTTTTTTGTCGACTTCACCAATGTCTGGATTCT-3′; SEQ.ID.NO:40) was employed toincorporate a SalI restriction site and a methionine initiation codonimmediately upstream of Ala², Ser⁶¹, Leu⁸⁰, and Phe¹²⁹. The plastidtargeting sequence (e.g., N-terminal sequence) was removed by sequentialdigest with SalI followed by NotI.

In specific embodiments, the Ala² mutant amino acid sequence(SEQ.ID.NO:33) is encoded by the nucleic acid sequence SEQ.ID.NO:32; theSer⁶¹ mutant amino acid sequence (SEQ.ID.NO:35) is encoded by thenucleic acid sequence of SEQ.ID.NO:34; the Leu⁸⁰ mutant amino acidsequence (SEQ.ID.NO:37) is encoded by the nucleic acid sequence ofSEQ.ID.NO:36; and the Phe¹²⁹ mutant amino acid sequence (SEQ.ID.NO:39)is encoded by the nucleic acid sequence of SEQ.ID.NO:38. In a specificembodiment, an N-terminal truncation at any point in the amino acidsequence up to and including amino acid 129. In specific embodiments,alternative truncations are generated at the following sites: Cys⁵⁵,Glu⁷⁴, Glu⁷⁶, or Val⁸⁸, wherein the truncation site occurs just prior tothe indicated amino acid (for example, between Asn⁵⁴ and Cys⁵⁵). Askilled artisan is aware that sequences having N-terminal truncationspreferably have an ATG start codon included.

A. Expression in a Prokaryote

The desired plasmids were prepared by ligating the mutated gene insertinto the similarly digested vectors pET32c(+) (Novagen; Madison, Wis.)and pRS426GAL (Hua, 2000), a multiple copy yeast expression vector.These plasmids was expressed in E. coli BL21(DE3) (Novagen; Madison,Wis.). E. coli cells were grown in Luria-Bertani medium supplementedwith 100 μg/mL ampicillin at 37° C. with shaking to OD₆₀₀ ˜0.6. Thefollowing parameters were tested: isopropyl 1-thio-β-D-galactopyranoside(IPTG) concentration (50, 100, 250, 500, and 1000 μM); and temperatureand time (20° C. for 2, 3, 4, 6, 21 hours, 22° C. for 6, 8, 16, 19, 22,45 hours, and 30° C. for 3, 6 hours). The following assay conditionswere tested to obtain maximum diterpene product yield: 30 mM HEPES(N-2-hydroxyethylpiperazine-N′-4-butanesulfonic acid), pH 6.9, 7.2, 7.6,8.0; 30 mM Tris (tris(hydroxymethyl)aminomethane hydrochloride), pH 7.4,7.8, 8.2; 1, 5, 10% glycerol; 1, 3, 5, 10, 20 mM DTT (dithiothreitol);20 mM β-mercaptoethanol; 2, 5, 8% Triton X-100; 5% Tween 80; 0, 2, 7.5,20, 50 mM MgCl₂; 0, 30, 500, 1000 μM MnCl₂; 2, 10, 13.3, 20, 40, 80, 200μM GGDP; and 23° C. and 32° C. assay temperatures.

Optimal soluble protein production and diterpene yield were obtainedwith the following conditions. Cell cultures were induced with 1 mM IPTGat 20° C. with shaking for 6 hours and lysed by sonication in 30 mMHEPES, pH 7.2, 5 mM DTT, and 5% glycerol. The soluble fraction of thelysate (100 mg/mL) was incubated with 20 μM GGDP in 30 mM HEPES, pH 7.2,5 mM DTT, 5% glycerol, 2 mM MgCl₂, and 500 μM MnCl₂ overnight at 32° C.

Levopimaradiene synthase was also tested for activity towards 200 μMgeranyl diphosphate and farnesyl diphosphate. Cell cultures were inducedand lysed as noted above. The soluble fraction of the lysate wasincubated overnight at 32° C. with 200 μM substrate in 30 mM HEPES, pH7.2, 5 mM DTT, 5% glycerol, 2 mM MgCl₂ and 500 μM MnCl₂, and overlaidwith hexane (1 mL).

B. Expression in a Eukaryote

Expression in S. cerevisiae JBY575, which represents wild-type yeast,was observed. JBY575 cells transformed with pRS426GAL inserted with theputative levopimaradiene synthase were grown in synthetic completemedium lacking uracil and supplemented with 2% glucose and 1.5% agar at30° C. to saturation and induced with galactose for 48 hours. Cells wereharvested, resuspended in lysis buffer, and mixed by vortexing overglass beads. The lysate was assayed with 60 μM GGDP in the presence andabsence of 0.2% and 5% Triton X-100.

All in vitro reactions were extracted 3× with hexane and dried overMgSO₄. The reaction was further extracted twice with diethyl ether anddried over MgSO₄. Thereafter, the crude lysate was suspended in 100 mMTris, pH 8.0 containing 2.9 units/mg apyrase (a dephosphorylating agent)and 10 units/μL calf intestinal alkaline phosphatase, incubated at 30°C. for 3 hours, and extracted with diethyl ether as noted above (Croteauand Cane, 1985). The crude reaction mixtures were eluted over SiO₂,concentrated, and analyzed by GC and GC/MS.

Gas chromatography spectra were obtained on a Hewlett Packard 6890Series GC System equipped with an Rt_(x)-5 capillary column (Restek, 30m×0.25 mm i.d., 0.10 μm d_(f)). The following separation conditions andtemperature program were employed: injector port 250° C., FID 250° C.,split ratio 39:1, helium flow 20 cm/s (0.6 mL/min), 150° C. hold 5 min,increase to 250° C. (5° C./min), hold 5 min. GC/MS spectra were obtainedon a Hewlett-Packard 5890A instrument with a 30-m DB-5 ms column (J&WScientific Inc., 0.25 mm i.d., 0.10 μm df). The following separationconditions and temperature program were employed: injector port 280° C.,transfer lines: 280° C., helium flow at 30 cm/s (1 mL/min) withsplitless injection at 150° C. hold 3 min, increase to 250° C. (5° C./min), hold 5 min. Mass spectra (m/z 50 to 500) were obtained on a VGZAB-HF reverse-geometry double-focusing instrument at 70 eV with anelectron-impact ion source (200° C.). Accelerating voltage was set to 8kV and the resolution was 1000 (10% valley).

Example 5 Levopimaradiene Standard-Synthesis and Structural Confirmation

Levopimarol (95.0 mg, 0.33 mmol) was dissolved in 3.7 mL dichloromethaneand 92 μL triethylamine then cooled to 0° C. Methanesulfonyl chloride(31 μL, 0.39 mmol) was added dropwise via syringe. The reaction wasmonitored by thin layer chromatography (TLC) (1:1 chloroform:diethylether) and quenched after 15 min with ice-cold saturated aqueous sodiumbicarbonate. The solution was extracted with dichloromethane (3×),washed with H₂O, dried with MgSO₄, filtered, and concentrated. (Cambieet al., 1990) The crude material was purified by preparative TLC (1 mmSiO₂, 1:1 chloroform:diethyl ether) yielding 75.1 mglevo-8,12-dien-18-yl methanesulfonate (62.4% yield, R_(f)0.96). ¹H NMR(CDCl₃, 400 MHz) δ 5.54 (q, J=1.8 Hz, 1H, H-14), 5.15 (t, J=4.3 Hz, 1H,H-12), 3.97 (d, J=9.4 Hz, 1H, H-18), 3.73 (d, J=9.4 Hz, 1H, H-18), 3.00(s, 3H, CH₃SO₂), 2.38-2.28 (m, 3H, H-7α, H-11α, H-11α), 2.19-2.03 (m,3H), 1.76 (dt, 1H), 1.61-1.52 (m, 3H), 1.47-1.34 (m, 4H), 1.27-1.21 (m,1H), 0.97 (d, 6H, H-16, H-17), 0.91 (s, 3H), 0.88 (s, 3H).

Under an inert atmosphere, the mesylate (21.5 mg, 0.06 mmol) wasdissolved in tetrahydrofuran in a Schlenk flask equipped with a coldfinger. Excess lithium triethylborohydride (263 μL, 1 M intetrahydrofuran, 0.26 mmol) was added dropwise to the solution, thereaction was stirred at reflux for 6 hours and monitored by TLC (6:1hexane:diethyl ether). The reaction was quenched with ice-cold H2O,extracted with hexane (3×), dried with MgSO₄, filtered over a silicaplug, and concentrated. (Walter, 1988) GC analysis indicated that an85:15 mixture of levopimaradiene:abietatriene had been obtained in 41.3%yield (6.6 mg). The isomeric mixture was separated by argentic TLC (Liet al., 1995) (SiO₂-AgNO₃, 3 developments with 85:15 hexane:diethylether) giving pure abietatriene (R_(f) 0.96) (Kutney and Han, 1996) andpure levopimaradiene (R_(f) 0.92) as identified by de novocharacterization based on ¹H, COSY-DEC, ¹³C, DEPT-135, HSQC, and HMBCNMR, and GC/MS analyses.

¹H NMR (CDCl₃, 500 MHz, 25° C.) δ 5.518 (q, J=1.8 Hz, 1H, H-14), 5.141(br tq, J=4.3, 1.3 Hz, 1H, H-12), 2.338 (ddd, J=13.3, 4.5, 2.2 Hz, 1H,H-7β), 2.323, 2.307 (m, 2H, H-11α, H-11β), 2.145 (septet of q, J=6.8,1.3 Hz, 1H, H-15), 2.075 (br td, J=13.2, 5.2 Hz, 1H, H-7α), 2.021 (ddt,J=11.5, 8.6, 1.8 Hz, 1H, H-9α), 1.737 (dtd, J=12.9, 3.4, 1.6 Hz, 1H,H-1β), 1.697 (ddt, J=12.7, 5.4, 2.7 Hz, 1H, H-6α), 1.519 (dt, J=13.5,3.4 Hz, 1H, H-2α), 1.444 (m, 1H, H-2β), 1.386 (m, 1H, H-3β), 1.368 (qd,J=12.8, 4.5 Hz, 1H, H-6β), 1.149 (tdd, J=13.2, ˜4.0, 0.8 Hz, 1H, H-3α),1.045 (dd, J=12.5, 2.8 Hz, 1H, H-5α), 0.975 (d, J=6.8 Hz, 6H, H-16,H-17), 0.866 (td, J=˜12.7, ˜3.4 Hz, 1H, H-1α), 0.862 (s, 3H, H-18),0.861 (s, 3H, H-20), 0.821 (s, 3H, H-19). Chemical shifts werereferenced to Si(CH₃)₄ and are accurate to ±0.001. Coupling constantsare accurate to ±0.5 Hz.

¹³C NMR (CDCl₃, 125 MHz, 25° C.) δ 139.46 (C-8), 138.91 (C-13), 118.73(C-14), 114.87 (C-12), 55.23 (C-5), 49.61 (C-9), 42.21 (C-3), 40.75(C-10), 37.91 (C-1), 36.15 (C-7), 33.48 (C-20), 33.45 (C-4), 33.26(C-15), 23.80 (C-6), 22.75 (C-11), 21.80 (C-19), 21.45 (C-17), 21.37(C-16), 19.00 (C-2), 14.10 (C-18). Chemical shifts (±0.02 ppm) werereferenced to the CDCl₃ signal at 77.0 ppm.

GC/MS EI⁺ m/z (%)=272 [M⁺] (73), 257 [M−CH₃] (13), 229 [M−CH(CH₃)₂] (7),148 [M−C₉H₁₆] (64), 147 (27), 146 (50), 137 [M−C₁₀H₁₅] (94), 136 (65),135 (60), 134 (90), 133 (66), 131 (43), 119 (27), 117 (34), 105 (58), 95(31), 93 (28), 92 (100), 91 [M−C₁₃H₂₅] (97), 83 (20), 81 (26), 69 (24).GC co-elution of synthetic levopimaradiene with the enzymatic productand GC/MS fragmentation confirmed the identity of the in vitro productditerpene as levopimaradiene.

Example 6 Isolation and Characterization of a Diterpene Cyclase cDNAfrom Ginkgo Biloba

G. biloba cDNA libraries were prepared from cultivated embryonic roots.A homology-based approach utilizing PCR was employed to screen thelibrary. Degenerate primers were designed based on conserved sequenceregions among gymnosperm terpene synthases. These included Abies grandisabietadiene synthase, a bifunctional diterpene synthase that directsboth proton-initiated cyclization and ionization of the divalent metalcation-diphosphate ester moiety; and synthases that effect diphosphateionization to induce cyclization, including the diterpene Taxusbrevifolia taxadiene synthase; the sesquiterpenes Abies grandisbisabolene synthase, selinene synthase, and humulene synthase; and themonoterpenes Abies grandis pinene synthase, limonene synthase, andmyrcene synthase. Seven forward and eight reverse degenerate primersidentifying eight regions of high sequence homology were designed. Thecombination of HSG1FP with HSG6RP resulted in amplification of a 1907 bpfragment (HSG97), which was determined to have significant sequencehomology to higher plant terpene cyclases. A segment of this fragmentwas ³²P-labeled and used as a hybridization probe for high stringencyscreening of 3.0×10⁵ colonies from cDNA library HSG2. A skilled artisanis aware that the cDNA preferably comprises a majority of expressedsequences, which are also preferably full-length, from an organism.

A total of 10 hybridizing colonies were obtained and put through asecondary round of high stringency screening producing an enriched poolof clones. The termini of the three longest cDNAs were sequenced andidentified as putative diterpene cyclases based on homology to Abiesgrandis abietadiene synthase. Furthermore, all three clones hadidentical 5′ and 3′ ends (approximately 600 bp at each end). One clonewas further characterized. Sequence data revealed that a 2681 bp cDNAhad been cloned, however, the initiation codon was absent. RNA ligasemediated rapid amplification of cDNA ends (“RACE”) was employed toisolate the 5′-untranslated region and the methionine start site. Thefull-length gene, Ginkgo biloba levopimaradiene synthase, a diterpenesynthase, was 2705 bp in length and encoded an 873 amino acid openreading frame of 2622 bp with a predicted molecular weight of 100,289(see FIG. 4).

Example 7 Analysis and Selection of Plastid Targeting Sequence CleavageSite

Cytosolically synthesized plastid proteins contain N-terminal targetingsequences that direct their translocation to specific plastidialcompartments. Proteolysis of the signal sequence occurs by a specificprotease, yielding the mature protein. Plastid transit peptidestypically range between 30 to 80 amino acids in length; are rich inhydroxlated amino acids, basic amino acids, and small hydrophobicresidues; and display low contents of tyrosine and acidic residues. Forpurposes of heterologous expression, wherein native processingpeptidases are not present, cleavage of the signal sequence may berequired prior to expression to avoid formation of inclusion bodies. Ingeneral, cleavage sites are distinguished by a decreased frequency ofserine residues and a corresponding increase in the frequency oftyrosine and acidic amino acids. In a majority of higher eukaryotes,arginine is found at positions −2 and −6 to −10 relative to the cleavagesite. Furthermore, a consensus motif of (Val/Ile)-X-(Ala/Cys)↓Ala(wherein the downward arrow (↓) indicates the site of bond cleavage) hasbeen identified in a series of stroma-targeting chloroplast transitpeptides with known cleavage sites (von Heijne and Nishikawa, 1991; vonHeijne and Gavel, 1990; Keegstra and Olsen, 1989).

Analysis of Ginkgo biloba levopimaradiene synthase indicated thefollowing representation of amino acid residues: the first tyrosineresidue at Y⁸⁴; the first glutamic acid residue at E⁶⁴; the firstaspartic acid residue at D68; and a decreased frequency of serineresidues between S⁴⁷ and S⁹⁶. Two potential cleavage sites wereidentified at Ile-His-Ala⁶⁰↓Ser⁶¹ (with arginine at −9 and −11 relativeto the cleavage site) and at Ile-Gln-Cys¹²⁷↓Met¹²⁸ (with arginine at −11relative to the cleavage site). Submission of Ginkgo bilobalevopimaradiene synthase to META Predict Protein Chloro P predicted thepresence of an N-terminal chloroplast transit peptide with a cleavagesite between H⁵⁹-A⁶⁰ (Nielsen et al., 1995). Three truncation sites wereselected in consideration of the data presented above: Ala⁶⁰-Ser⁶¹(hereafter referred to as Ser⁶¹), Arg⁷⁹-Leu⁸⁰ (hereafter referred to asLeu⁸⁰), and Cys¹²⁷-Met¹²⁸ (hereafter referred to as Phe¹²⁹).

Recently, successful heterologous expression of truncatedlevopimaradiene synthases have been reported. Cleavage of the N-terminal84 residues of Abies grandis abietadiene synthase produced activeprotein (Ravn et al., 2000). Truncation of 79 or fewer residues of Taxusbrevifolia taxadiene synthase produced functional protein, however,elimination of 93 or more residues resulted in loss of catalyticactivity (Williams et al., 2000). Low primary sequence homology isobserved between Ginkgo biloba levopimaradiene synthase, Abies grandisabietadiene synthase, and Taxus brevifolia taxadiene synthase prior toresidue Ginkgo biloba levopimaradiene synthase Trp⁸⁹, 21.5% and 14.0%,respectively. However, significant sequence similarity begins atposition Trp⁸⁹, 65.7% and 44.1% respectively. Furthermore, no distinctidentity is apparent between these synthases at the truncation sitesreported to produce functional protein.

Example 8 Levopimaradiene Synthase Sequence Analysis

Protein analysis of the deduced polypeptide indicated Ginkgo bilobalevopimaradiene synthase to have high sequence similarity to Abiesgrandis abietadiene synthase (60%), Abies grandis bisabolene synthase(46%), and Taxus brevifolia taxadiene synthase (41%). Threeaspartate-rich motifs and a putative plastidial transit peptide wereidentified in Ginkgo biloba levopimaradiene synthase. An N-terminalDDXID motif (Ginkgo biloba levopimaradiene synthase 91-95), alsoobserved in Abies grandis abietadiene synthase, may serve to stabilizecarbocations and/or direct deprotonation. Crystallographic andmutagenesis studies suggest that the consensus motif, D(I/V)DDTA (Ginkgobiloba levopimaradiene synthase 405-410), initiates cyclization of GGDP.Moreover, this aspartate-rich sequence remains highly conserved amongsynthases that effect proton-initiated cyclization, including copalyldiphosphate synthases, Abies grandis abietadiene synthase, Phaeosphaeriaent-kaurene synthase, and squalene-hopene cyclases (Bohlman et al.,1998). A carboxy-terminal DDXXD motif (Ginkgo biloba levopimaradienesynthase 624-628) resides in kaurene synthases; Abies grandisabietadiene synthase; prenyltransferases; and in plant mono-, sesqui-,and diterpene synthases (Bohlman et al., 1998). This domain in aspecific embodiment affects binding of the divalent metalion-diphosphate complex. Crystal structure analysis of tobaccoepi-aristolochene synthase identified two Mg²⁺ ions bound at theentrance of the active site by coordination to aspartic acid residues ofthe DDXXD.motif (Starks et al., 1997).

Comparative protein analysis indicated that Ginkgo bilobalevopimaradiene synthase contained features reminiscent of two distinctcatalytic domains, and thereby confirmed it as a bifunctionallevopimaradiene synthase. Furthermore, Ginkgo biloba levopimaradienesynthase displayed a high degree of homology to conserved amino acidresidues of mono-, sesqui-, and diterpene secondary metabolite families(Bohlman et al., 1997). These included the absolutely or highlyconserved residues Ser⁴⁵⁹, Ala⁴⁷², Pro⁷¹³, Cys⁷⁸⁹,Arg^(414, 417, 587, 610, 766); the acidic residuesAsp^(363, 624, 625, 628, 770, 851) and Glu^(466, 567, 592, 703, 717);and the aromatic residues His⁴¹⁹, Phe^(431, 438, 585, 663),Tyr^(523, 594, 700, 847), and Trp^(89, 574, 646, 706). Three significantdeviations in the Ginkgo biloba levopimaradiene synthase sequenceincluded a highly conserved histidine which corresponds to Ginkgo bilobalevopimaradiene synthase Tyr³⁷³, an absolutely conserved proline whichcorresponds to Ginkgo biloba levopimaradiene synthase Arg⁶⁵⁵, and anabsolutely conserved acidic amino acid which corresponds to Ginkgobiloba levopimaradiene synthase Gly⁶⁷².

Example 9 Protein Expression and Optimization of In Vitro EnzymaticActivity

Site-directed mutagenesis was employed to insert a SalI site followed byseven adenines and a methionine start codon at Ala², Ser⁶¹, Leu⁸⁰, andPhe¹²⁹. Following removal of the 5′-untranslated region and plastidtargeting sequence, the desired plasmids were prepared by ligation withpET32c(+) (a bacterial expression system containing a thioredoxin tagdesigned for maximal production of soluble protein) and pRS426GAL (amultiple copy yeast expression system). The levopimaradiene synthase wasexpressed in the E. coli strain BL21(DE3) and the wild-type S.cerevisiae strain JBY575, respectively. E. coli cells were grown inLuria-Bertani medium supplemented with ampicillin and induced with IPTG.SDS-PAGE analysis indicated that protein production increased with timeand reached maximum accumulation by 21 hours. However, recombinantprotein resided mainly in the insoluble fractions of the lysate,indicating that it was likely encapsulated in an inclusion body.Attempts to improve protein solubility by variation of IPTGconcentrations between 50 to 1000 mM were unsuccessful. However,employing an induction temperature of 20° C. produced functionallysoluble protein.

The skilled artisan recognizes that lysis and assay conditions should beoptimized. Examples of parameters that can be adjusted to optimizeconditions include altering buffer, pH, reductant, reductantconcentration, metal cofactors, cofactor concentrations, glycerolconcentrations, substrate concentrations, and assay temperatures.Levopimaradiene synthase activity proved to be independent of magnesiumbut required manganese cofactor for catalysis. Maximum soluble proteinproduction and diterpene yield were obtained with the followingconditions. Cell cultures were induced with 1 mM IPTG at 20° C. withshaking for 6 hours and lysed by sonication in 30 mM HEPES, pH 7.2, 5 mMDTT, and 5% glycerol. The soluble fraction of the lysate (100 mg/1 mL)was incubated with 20 μM GGDP in 30 mM HEPES, pH 7.2, 5 mM DTT, 5%glycerol, 2 mM MgCl₂, and 500 μM MnCl₂ overnight at 32° C. Yeastexpression was induced with galactose at 30° C. for 48 hours and thecells were lysed and assayed according to the conditions noted.

The extent of N-terminal truncation affected catalytic activity in bothexpression hosts. Bacterial expression of Ginkgo biloba levopimaradienesynthase truncated at Ala², Ser⁶¹, and Leu⁸⁰ produced levopimaradiene asthe exclusive diterpene hydrocarbon, however, Phe¹²⁹ failed to producedetectable levels of any diterpene. Highest expression levels wereobtained with the Ser⁶¹ truncation (approximately 1% turnover of GGDP)and lowest levels were obtained with Ala², with approximately 80%difference in activity. Yeast expression of Ser⁶¹ and Leu⁸⁰ yieldedlevopimaradiene as the sole diterpene. However, both the Ala² and Phe¹²⁹truncated genes failed to produce observable levels of any diterpeneproduct. Controls performed in parallel did not yield levopimaradiene.E. coli expression and incubation with geranyl diphosphate and farnesyldiphosphate did not produce any identifiable terpenes by GC or GC/MS.However, a skilled artisan is aware of parameters which may be optimizedand/or additional sequences which may be employed to detect and/orincrease synthesis of levopimaradiene synthase.

Example 10 Product Characterization

Due to the low levels of levopimaradiene production, a syntheticstandard to confirm product identification was utilized. Levopimaricacid was obtained from Helix Biotech and converted to levopimarolaccording to literature procedures, with care employed to minimizeexposure to oxygen and heat. Reaction of the alcohol with mesyl chlorideand triethylamine followed by silica gel purification resulted in a 62%yield of the mesylate derivative. The ester was reduced withSuper-Hydride® to yield a 41% mixture of levopimaradiene:abietatriene(85:15). Argentic chromatography effected the separation of thehydrocarbons; levopimaradiene was identified by NMR and GC/MS analysis.Co-elution on GC and identical GC/MS fragmentation of the biosynthetichydrocarbon with the synthetically prepared levopimaradiene confirmedidentification of the enzyme product to be levopimaradiene.

Molecular biology techniques were employed to confirm the presence oflevopimaradiene as a rapidly metabolized intermediate. The bifunctionalenzyme directs a multi-step mechanistic sequence in which GGDP iscyclized to labdadienyl diphosphate which undergoes allylic ionizationof the ester moiety followed by hydrogen shift, methyl migration, anddeprotonation yielding levopimaradiene.

Example 11 Expression In Vivo in Yeast that Accumulate GGDP

A skilled artisan recognizes that it is preferable to increase theamount of substrate provided for in the production of levopimaradiene toultimately increase ginkgolide yields. Thus, the invention preferablyincludes an increase in the amount of effective geranylgeranyldiphosphate, which is upstream in the ginkgolide biosynthetic pathway. Askilled artisan recognizes that an effective amount of geranylgeranyldiphosphate is that which is subject to metabolism in the isoprenebiosynthetic pathway. A skilled artisan is aware that an increase inGGDP occurs in multiple ways, such as by providing GGDP exogenously orby increasing its production through transgenic and/or bioengineeringmeans. GGDP is increased by the methods and compositions of theinvention described in the U.S. patent application entitled,“Diterpene-Producing Unicellular Organism” filed on the same day andincorporated by reference herein.

The yeast strain of the copending application employed was EHY18 (Hart,E., 2001) which was further transformed with multiple-copy yeastexpression vectors comprising an isolated and purified nucleic acidsequence of GbLS or derivatives thereof, which are described hereinunder control of the GAL1 inducible promoter. Yeast cells were grown tosaturation in 5 mL ScD-Leu-Ura at 30° C. Cells were harvested (1300×g, 2min, 25° C.) and resuspended in 500 μL sterile Milli-Q H₂O (2×). Thewashed cells were resuspended in 5 mL sterile Milli-Q H₂O. An aliquot ofthe culture (5 μL) was added to a 25 mL Corex tube containingpre-prepared 2× resin and 5 mL ScG-Leu-Ura (4% galactose) and shaken at30° C. for 6 days. Resin was collected by filtration through a Konteschromatography column (1.7 cm diameter) and rinsed with dI H₂O untileluent was clear. The resin was incubated in 2 mL ethanol for severalminutes, eluted, and repeated 2×. The combined eluents were dissolved inapproximately 3 mL dI H₂O and extracted (3×) with 3 mL hexane. Thecombined extracts were concentrated under a stream of nitrogen. GCanalysis (quantitation extrapolated from 0.2 mg/mL longifolene externalstandard) indicated the following product profile (obtained fromtriplicate measurement of three cultures): 0.61±0.20 mg/Llevopimaradiene, 0.04±0.01 mg/L abietadiene, 0.15±0.05 mg/Labietatriene, 0.16±0.13 mg/L (+)-copalol, and 1.79±0.90 mg/Lgeranylgeraniol. GC/MS fragmentation confirmed the identity of eachcompound.

A large scale culture (1-L) was prepared by growing the yeast cells tosaturation in 5 mL ScD-Leu-Ura at 30 ° C. Cells were harvested (1300×g,2 min, 25° C.) and resuspended in 500 μL sterile Milli-Q H₂O (2×). Thewashed cells were resuspended in 5 mL sterile Milli-Q H₂O. An aliquot ofthe culture (1 mL) was added to a 2 L flask containing 1-L ScG-Leu-Ura(4% galactose) and 88.27 g resin in 120 mL Milli-Q H₂O (pre-prepared).The resin was eluted with ethanol (3×100 mL with 20 min incubationperiods), which was subsequently extracted (6×) with hexane. Thecombined extracts were dried over magnesium sulfate, filtered, andconcentrated under reduced pressure. GC analysis (quantitationextrapolated from 0.2 mg/mL longifolene external standard) indicated thefollowing product profile (obtained from triplicate measurement):0.29±0.04 mg/L levopimaradiene, 0.04±0.02 mg/L abietadiene, 0.13±0.04mg/L abietatriene, 011±0.02 mg/L (+)-copalol, and 0.56±0.03 mg/Lgeranylgeraniol. GC/MS analyses confirmed the production of the abovenoted diterpenes, which included hydrocarbons and an alcohol.

Example 12 Optimization of Levopimaradiene Production

Similar to levopimaradiene synthase activity in E. coli, catalyticactivity in EHY18 was affected by the N-terminal truncation of GbLS.Expression of wild-type levopimaradiene synthase and its truncatedcounterparts Ser61 and Leu80 produced (+)-copalol, levopimaradiene,abietadiene, and abietatriene. Co-elution on GC and identical GC/MSfragmentation of the biosynthetic alcohol with synthetic (+)-copalolallowed the unequivocal identification of the enzyme product as(+)-copalol. GC analysis of the expressed GbLS Phe129 indicated noditerpene alcohol or hydrocarbon formation. Highest diterpene productionwas obtained with the Ser61 and Leu80 truncated constructs (Ser61≧Leu80)and was approximately four times greater than that observed for thewild-type synthase. Negative controls performed in parallel (expressingEHY18) did not yield levopimaradiene, abietadiene, abietatriene, or(+)-copalol.

Further optimization studies employed the Ser61 construct. Factorsinfluencing diterpene production included induction period andconcentration of galactose and resin in the inducing medium. Highestditerpene yields were observed with 4% galactose and 0.70 g resin/5 mLculture medium. Expression for 6 days, in the above noted inductionmedium, resulted in a net yield of ˜0.8 mg/L diterpene hydrocarbons and˜0.2 mg/L (+)-copalol, as determined by GC quantitation.

Expression of GbLS in EHY18 resulted in a three- to six-fold increase inlevopimaradiene yield relative to the bacterial expression systempreviously employed. In addition, expression of GbLS in EHY18 affordedabietatriene, the immediate hydrocarbon precursor of the ginkgolides.The ability to enhance levopimaradiene production in a yeast cell havingan increased effective amount of geranylgeranyl diphosphate suggests thesystem is useful in the isolation of the first oxygenase involved inginkgolide biosynthesis. Lastly, identification of (+)-copalolrepresents the first observation of the free intermediate of (+)-copalylpyrophosphate by a bifunctional diterpene catalyst and supports previousdata implicating (+)-copalyl pyrophosphate as a precursor tolevopimaradiene (Peters et al., 2000; Schwarz and Arigoni, 1999).

Bacterial expression optimization includes standard manipulations knownin the art to overcome problems such as low solubility and lowexpression levels. For instance, a skilled artisan is aware thatdifferent E. coli expression systems, including commercially availablevectors and strains, are utilized to produce higher amounts of solubleprotein. An example of a vector includes pSBET (Schenk et al., 1995),which is particularly useful for heterologous expression in Escherichiacoli of plant genes that often have a significant number of arginineresidues. The vector is particularly well-suited to Escherichia coliBL21 (DE3) (Sambrook et al., 1989). Also, many different E. coli strainsare known in the art and may be used, such as LE392 cells, DH5α cells,or SURE™ (Stratagene; La Jolla, Calif.) cells.

Example 13 Cloning Ginkgolide Biosynthetic Genes

Difficulties in RNA extraction from recalcitrant gymnosperm tissue havebeen noted (Chang, et al., 1993;Lewinsohn, et al., 1994). High levels ofpolysaccharides in gymnosperm tissue and oxidation of polyphenols duringextraction resulted in contaminated and/or degraded RNA. With respect toG. biloba tissue, studies indicate that mature ginkgo seeds arecomprised of approximately 35% water-soluble polysaccharides (Arahira,et al., 1994). However, successful construction of a Ginkgo biloba cDNAlibrary, as described herein, has overcome this problem.

In a specific embodiment, nucleic acid sequences encoding other enzymesin the ginkgolide biosynthesis pathway are obtained. In a specificembodiment, a cDNA library, such as for E. coli or S. cerevisiae,comprising Ginkgo biloba sequences are exposed to an E. coli or S.cerevisiae cell, respectively, wherein the cell also comprises thelevopimaradiene synthase sequence, and the presence of a desireddownstream product is assayed. In a specific embodiment, the GC and/orGC/MS profile of the product is known and its presence is determined. Ina further specific example, the nucleic acid sequence for adehydrogenase, which generates formation of abietatriene, is cloned byassaying pools of cells harboring levopimaradiene synthase and.identifying by chromatography (i.e., GC or GC/MS) the pool in whichabietatriene is produced. Once a pool is identified, this pool is brokendown into its constituents which are assayed in smaller pools and/orindividually to identify the cell containing the clone expressing thedesired nucleic acid sequence.

In an embodiment of the present invention, a first ginkgolidebiosynthetic gene downstream of levopimaradiene synthase is provided ina cell comprising the levopimaradiene synthase, wherein both the firstdownstream gene and the levopimaradiene synthase are expressedconcomitantly. In a specific embodiment, the cell provides biosynthesisof a ginkgolide biosynthetic intermediate that is a first derivative oflevopimaradiene, such as abietatriene. A further embodiment is thesubsequent cloning of a second downstream ginkgolide biosynthetic gene,which allows biosynthesis of a different ginkgolide biosyntheticintermediate upon expression in a cell comprising the levopimaradienesynthase, the first downstream ginkgolide biosynthetic gene and thesecond ginkgolide biosynthetic gene; this cell demonstrates biosynthesisof a ginkgolide biosynthetic intermediate that is a second derivative ofthe first derivative (e.g., abietatriene) of levopimaradiene. Other suchembodiments are contemplated in which levopimaradiene, produced by acell that expresses the Ginkgo biloba levopimaradiene synthase andconservatively modified variants, serves as an intermediate inbiosynthesis of a diterpenoid, and preferably a ginkgolide.

Example 14 Summary of the Present Invention

Levopimaradiene synthase, which directs the first committed step inginkgolide biosynthesis, was cloned and functionally characterized aspart of a program to isolate and express genes involved in thebiosynthesis of the ginkgolides. A Ginkgo biloba cDNA library wasprepared from embryonic roots and screened utilizing a homology-basedapproach employing degenerate primers with high sequence similarity togymnosperm terpene synthases. Polymerase chain reaction amplificationprovided a 1907 bp fragment, which was employed to probe the library.Colony hybridization and rapid amplification of cDNA ends yielded afull-length clone with a 2622 bp open reading frame encoding a predictedprotein sequence of 873 amino acids with an estimated molecular weightof 100,289. Protein analysis indicated that a bifunctional terpenecyclase had been isolated with high sequence identity to Abies grandisabietadiene synthase (60%), Abies grandis bisabolene synthase (46%), andTaxus brevifolia taxadiene synthase (41%). Additionally, the amino acidsequence contained a putative N-terminal plastidial transit peptide andthree aspartate-rich regions.

Functional expression in Escherichia coli of the full-length cDNA andcorresponding truncations at Ser⁶¹ and Leu⁸⁰ provided enzymatic activitycapable of cyclizing geranylgeranyl diphosphate to levopimaradiene, asconfirmed by GC/MS analysis. Expression of the truncated Phe¹²⁹ geneproduct resulted in complete loss of enzymatic activity. Functionalexpression in wild-type Saccharomyces cerevisiae of the Ser⁶¹ and Leu⁸⁰truncations yielded levopimaradiene synthase activity, albeit in loweryields than with the bacterial system, whereas the full-length andPhe¹²⁹ clones failed to produce detectable levels of biosyntheticproduct. Isolation and characterization of levopimaradiene synthaserepresents the first confirmation of an enzyme involved in ginkgolidebiosynthesis.

An engineered yeast strain has been employed to achieve increasedlevopimaradiene production levels. An approximate three-fold to six-foldincrease in levopimaradiene yield was obtained relative to thepreviously employed bacterial and yeast expression systems. In addition,production of abietatriene, the direct hydrocarbon progenitor of theginkgolides, was realized.

REFERENCES

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by referenceherein.

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One skilled in the art readily appreciates that the patent invention iswell adapted to carry out the objectives and obtain the ends andadvantages mentioned as well as those inherent therein. Plant cells,yeast cells, cell cultures, plants, sequences, methods, procedures andtechniques described herein are presently representative of thepreferred embodiments and are intended to be exemplary and are notintended as limitations of the scope. Changes therein and other useswill occur to those skilled in the art which are encompassed within thespirit of the invention or defined by the scope of the pending claims.

1.-18. (canceled)
 19. An isolated polypeptide comprising an amino acidhaving a sequence of a Ginkeo biloba levopimaradiene synthase.
 20. Anisolated polypeptide comprising an amino acid having a sequence ofSEQ.ID.NO:37.
 21. An isolated polypeptide according to claim 20, furthercomprising an amino acid having a sequence of SEQ.ID.NO:35.
 22. Anisolated polypeptide according to claim 20, further comprising an aminoacid having a sequence of SEQ.ID.NO:
 33. 23.-67. (canceled)
 68. Anisolated polypeptide according to claim 20, further comprising an aminoacid having a sequence of SEQ.ID.NO:
 2. 69. An isolated polypeptideaccording to claim 19, wherein the polypeptide is operable to producelevopimaradiene in vitro.
 70. An isolated polypeptide according to claim19, wherein the polypeptide is operable to produce levopimaradiene invivo.
 71. An isolated polypeptide according to claim 19, wherein thepolypeptide is operable to convert gemaylgeranyl diphosphate tolevopimaradiene.
 72. An isolated polypeptide according to claim 70,wherein the polypeptide is operable to produce levopimaradiene in aeukaryote.
 73. An isolated polypeptide according to claim 72, whereinthe polypeptide is operable to produce levopimaradiene in Escherichiacoli.
 74. An isolated polypeptide according to claim 70, wherein thepolypeptide is operable to produce levopimaradiene in yeast.
 75. Anisolated polypeptide according to claim 70, wherein the polypeptide isoperable to produce levopimaradiene in Saccaromyces, Candida albicans,or Kluyveromyces lactis.
 76. An isolated polypeptide according to claim20, wherein the polypeptide is operable to produce levopimaradiene invitro.
 77. An isolated polypeptide according to claim 20, wherein thepolypeptide is operable to produce levopimaradiene in vivo.
 78. Anisolated polypeptide according to claim 20, wherein the polypeptide isoperable to convert gemaylgeranyl diphosphate to levopimaradiene.
 79. Anisolated polypeptide according to claim 77, wherein the polypeptide isoperable to produce levopimaradiene in a eukaryote.
 80. An isolatedpolypeptide according to claim 79, wherein the polypeptide is operableto produce levopimaradiene in Escherichia coli.
 81. An isolatedpolypeptide according to claim 77, wherein the polypeptide is operableto produce levopimaradiene in yeast.
 82. An isolated polypeptideaccording to claim 77, wherein the polypeptide is operable to producelevopimaradiene in Saccaromyces, Candida albicans, or Kluyveromyceslactis.
 83. An isolated polypeptide according to claim 19, wherein thepolypeptide comprises an amino acid having a sequence of a Ginkgo bilobalevopimaradiene synthase containing a deletion in the N-terminal region.